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Paclitaxel (ptx) is one of the first - line chemotherapeutics used to treat patients with breast, ovarian, nonsmall cell lung cancer, and advanced forms of kaposi s sarcoma . The mechanism involves interfering with the normal breakdown of microtubules during cell division . Successful application of ptx in the clinic has been limited by its poor water solubility and the systemic toxicity . Taxol is a cremophor el / ethanol formulation of ptx that has been used in the clinic . However, cremophor el can cause hyperactivity reactions, neuropathy, and other serious side effects . Thus, there is a need to develop an alternative delivery system for ptx . Various macromolecular delivery systems such as liposomes, dendrimers, and nanoparticles are under investigation, among which polymeric micelles have gained considerable attention owing to ease in preparation and their very small sizes (10100 nm). Recent studies have substantiated that sub-100 nm was critical for a delivery system to achieve effective tumor targeting . Our group has previously developed peg - fts as a dual - functional carrier for the delivery of poorly water - soluble anticancer drugs . This system was constructed by coupling two molecules of s - trans, trans - farnesylthiosalicylic acid (fts) to poly(ethylene glycol) (peg, mw = 5000) through an ester linkage (peg5k - fts2). Different from most reported delivery systems that use inert excipients, our system employ water - insoluble drug fts as the hydrophobic region of polymeric micelles . It can inhibit both oncogenically activated ras and growth factor receptor - mediated ras activation, resulting in the inhibition of ras - dependent tumor growth . Preliminary study showed that the antitumor activity of fts was well retained following coupling to peg5k . Furthermore, peg5k - fts2 readily formed small - sized micelles (2030 nm) that are effective in loading and delivering ptx . In vivo study demonstrated that the antitumor activity of the ptx - loaded peg5k - fts2 micelles was significantly higher than that of taxol . Recent studies from us and others have shown that the vitamin e - based micellar system could be significantly improved via modulating the peg motifs and the molar ratio of peg / vitamin e. in another study with peg - embelin system, we showed that a conjugate with two embelin molecules linked to peg was significantly more effective than the conjugate with one embelin molecule coupled to peg . This has prompted us to carry out a similar study with peg - fts system . Four peg - fts conjugates that vary in the molecular weight of peg (peg2k vs peg5k) and the molar ratio of peg / fts (1/2 vs 1/4) have been developed . We demonstrated that peg5k - fts4 formed the most stable mixed micelles with ptx among the four peg - fts conjugates . Furthermore, ptx formulated in peg5k - fts4 micelles was the most effective formulation in inhibiting the tumor growth in vivo . Phosphate buffered saline (dpbs) was purchased from lonza (md, u. s. A.). Poly(ethylene glycol) methyl ether (meo peg oh, mw = 2000, 5000 kda), dimethyl sulfoxide (dmso), succinate anhydride, diethanolamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt), trypsin - edta solution, triton x-100, and dulbecco s modified eagle s medium (dmem) were all purchased from sigma - aldrich (mo, u. s. A.). Fetal bovine serum (fbs) and penicillin n - hydroxysuccinimide (nhs) and dicyclohexylcarbodiimide (dcc) were purchased from alfa aesar (ma, u. s. A.). 4-(dimethylamino) pyridine (dmap) was purchased from calbiochem - novabiochem corporation (ca, u. s. A.). All cell lines were cultured in dmem containing 5% fbs and 1% penicillin - streptomycin at 37 c in a humidified 5% co2 atmosphere . Peg5k - fts4 was prepared via solution phase condensation reactions (figure 1). We started to synthesize two hydroxyl group terminated peg monomethyl ether (meo peg5k peg5k(oh)2 by a facile chemical reaction with succinic anhydride and dmap . To obtain four hydroxyl groups the polymer was precipitated and washed by ice - cold diethyl ether and ethanol twice, respectively, and concentrated under vacuum . Oh4, fts, dcc, and dmap were then dissolved in chloroform and allowed to react overnight at room temperature . The solution was filtered and precipitated in ice - cold diethyl ether and ethanol twice respectively, and concentrated under vacuum . The powder was then dissolved in water and filtered through a filter with a pore size of 0.22 m . Ptx (10 mm in chloroform) and four peg - fts conjugates (10 mm in chloroform) were mixed at various carrier / drug ratios . The organic solvent was removed by nitrogen flow to form a thin film of drug / carrier mixture . The film was dried under vacuum for 1 h to remove the remaining solvent . Dpbs was added to hydrate the thin film and the drug - loaded micelles were formed . Unincorporated ptx (precipitate) was removed by filtering through a syringe filter (pore size: 0.22 m). The particle size and zeta potential of micelles were measured by a zetasizer (dls) (zetasizer nano zs instrument, malvern, worcestershire, u. k.). The morphology and size distribution of drug - free or drug - loaded peg2k - fts2, peg2k - fts4, peg5k - fts2, and peg5k - fts4 micelles were observed using transmission electron microscopy (tem). The copper grid was immersed in a drop of sample solution and stained with 1% uranyl acetate . Imaging was performed at room temperature on jeol jem-1011 . The critical micelle concentrations (cmc) of four peg - fts micelles were determined by using pyrene as a fluorescence probe . Four peg - fts conjugates, peg2k - fts2, peg2k - fts4, peg5k - fts2, and peg5k - fts4, were prepared in chloroform at 1.2 mg / ml, and various amounts were added to nine separate vials . Then 10 l of 1.8 10 m of pyrene in chloroform was added to each vial and the solution was mixed well . The organic solvent was removed by oil pump, and then 3 ml of milli - q water was added to each vial . The final pyrene concentration was 6 10 m with the four peg - fts conjugate concentrations ranging from 0.0001 to 0.5 mg / ml . The vials were kept on a shaker for 24 h at 37 c to reach equilibrium before fluorescence measurement . The fluorescence intensities of samples were measured at the excitation wavelength of 334 nm and emission wavelength of 390 nm by synergy h1 hybrid multi - mode microplate reader (winooski, vt, u. s. A.). The cmc is determined from the threshold concentration, where the sharp increase in pyrene fluorescence intensity is observed . The ptx loading efficiency was quantified by high performance liquid chromatography (hplc) (alliance 2695 - 2998 system) as described previously . Drug loading capacity (dlc) and drug loading efficiency (dle) were calculated according to the following equation: the in vitro ptx release kinetics for the four peg - fts micelles was determined by a dialysis method according to our published protocol . Briefly, ptx loaded peg - fts micelles at a concentration of 0.5 mg ptx / ml were placed into a dialysis bag (mw cutoff 14000). The dialysis bag was incubated in 200 ml pbs containing 0.5% (w / v) tween 80 with gentle shaking at 37 c . The concentrations of ptx remaining in the dialysis bag at designated time points were measured by hplc . Hemolysis assay was performed using fresh blood collected through cardiac puncture from rats . Red blood cells (rbcs) were collected by centrifugation and washed with pbs three times . Then rbcs were diluted in pbs with a final concentration of 2% w / v . A total of 1 ml of diluted rbc suspension was mixed with different concentrations (0.2 and 1.0 mg / ml) of four peg - fts micelles and pei, respectively, and then incubated at 37 c in an incubator shaker for 4 h. the mixtures were centrifuged, and supernatants were transferred into a 96-well plate . The release of hemoglobin was determined at 540 nm absorbance using a microplate reader . Rbcs incubated with pbs and triton x-100 (2%) were used as the negative and positive controls, respectively . The percentage of hemolysis of rbcs was calculated as (odsample odnegative control)/(odpositive control odnegative control) 100% . The cytotoxicity of ptx formulated in peg2k - fts2, peg2k - fts4, peg5k - fts2, and peg5k - fts4 micelles was assessed with several cancer cell lines and compared to free ptx in dmso, respectively . Briefly, 4t1.2 (1000 cells / after 24 h of incubation in dmem with 5% fbs and 1% streptomycin penicillin, the old medium was removed and the cells were incubated for 72 h in the presence of indicated concentrations of ptx (free or formulated in four peg - fts micelles). A total of 100 l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) in dpbs (0.5 mg / ml) was added to each well and cells were further incubated for 2 h. mtt formazan was solubilized by dmso . The absorbance in each well was measured by a microplate reader with wavelength at 550 nm and reference wavelength at 630 nm . Cell viability was calculated as [(odtreat odblank)/(odcontrol odblank) 100%]. The cytotoxicity of peg2k - fts2, peg2k - fts4, peg5k - fts2, and peg5k - fts4 micelles alone was similarly tested in 4t1.2, mcf-7, and hct-116 cell line as described above . Ras protein expression level in hct-116 cells was evaluated by western blotting following our published method . Briefly, hct-116 cells with 6070% confluency in a 6-well plate were treated with four peg - fts conjugates for 20 h at a fts concentration of 40 m . Female balb / c mice, 46 weeks in age, were purchased from charles river (davis, ca, u. s. A.). All animals were housed under pathogen - free conditions according to aaalac guidelines . All animal - related experiments were performed in full compliance with institutional guidelines and approved by the animal use and care administrative advisory committee at the university of pittsburgh . A syngeneic murine breast cancer model (4t1.2) was used to examine the therapeutic effect of different formulations of ptx . A total of 2 10 4t1.2 cells in 200 l of pbs were inoculated s.c . At the right flank of female balb / c mice . Treatments were started when tumors in the mice reached a tumor volume of 50 mm and this day was designated as day 1 . On day 1, these mice were randomly divided into six groups (n = 5) and administered i.v . With pbs (control), taxol (10 mg ptx / kg), ptx - loaded peg2k - fts2, peg2k - fts4, peg5k - fts2, and peg5k - fts4 micelles (10 mg of ptx / kg), respectively on days 1, 3, 5, 8, 10, and 12 . Tumor sizes were measured with digital caliper three times a week and calculated by the formula: (l w)/2, where l is the longest and w is the shortest of the tumor diameters (mm). To compare between groups, relative tumor volume (rtv) was calculated at each measurement time point (where rtv equals to the tumor volume at a given time point divided by the tumor volume prior to first treatment). Mice were sacrificed before the tumor reached 2000 mm . To monitor the potential toxicity, the body weights of all mice from different groups were measured every three days . In addition, serum levels of transaminases (ast, alt) in the mice with different treatments were investigated at the completion of the study . In all statistical analysis, the significance level was set at a probability of p <0.05 . All results were reported as the mean standard deviation (sd). Unless otherwise indicated . Statistical analysis was performed by student s t test for two groups, and one - way anova for multiple groups, followed by newman keuls test if p <0.05 . Peg5k - fts4 conjugate, containing four molecules of fts coupled to one molecule of peg5k via a labile ester linkage, was developed by solution phase condensation reactions . H nmr spectra of peg5k - fts4 conjugate are shown in supporting information figure s1d . The intense peak at 3.66 ppm was attributable to the methylene protons of peg . Carbon chain signals and benzene ring signals of fts were located at 1.52.2 ppm and 78 ppm, respectively . Maldi - tof suggested that four fts were successfully attached to peg5k (supporting information figure s2d). We also synthesized peg2k - fts2, peg2k - fts4, and peg5k - fts2 conjugates, which were confirmed by h nmr spectra and maldi the four peg - fts conjugates readily formed micelles in aqueous solution with the particle sizes of 2030 nm (table 1). Dynamic light scattering (dls) measurements showed that peg5k - fts4 micelles had hydrodynamic sizes around 27 nm at the concentration of 20 mg / ml (figure 2a). The size observed by tem shows good agreement with that determined by dls (supporting information figures s3 and s4). The spherical shape and size distribution were well retained when ptx was loaded into micelles at a drug concentration of 1 mg / ml and a carrier / drug ratio of 2.5/1 (m / m) (figure 2c and figure 2d). Similar results were shown for the other three micelles (supporting information figures s3 and s4). Particle size distribution and morphology of drug - free and ptx - loaded peg5k - fts4 micelles . The size and size distribution of drug - free peg5k - fts4 micelles (a) and ptx - loaded peg5k - fts4 micelles (c) were evaluated by dls . The morphology of drug - free peg5k - fts4 micelles (b) and ptx - loaded peg5k - fts4 micelles (d) was examined by tem . The cmc of peg2k - fts2, peg2k - fts4, peg5k - fts2, and peg5k - fts4 micelles were measured using pyrene as a fluorescence probe (table 1). When the concentration of the peg - fts reached the cmc, the fluorescence intensity of pyrene would change dramatically due to the transfer of pyrene from polar microenvironment to nonpolar surroundings caused by the formation of micelles . The cmcs of peg5k - fts2 and peg5k - fts4 conjugates were determined to be 0.34 and 0.29 m, respectively, which were lower than those of peg2k - fts2 (1.22 m) and peg2k - fts4 (1.43 m) (supporting information figure s5). The lower cmcs of peg5k - fts micelles may be attributed to the longer peg hydrophilic chain and more effective stabilizing effect for the micelles . The ptx loading of peg2k - fts2, peg2k - fts4, peg5k - fts2, and peg5k - fts4 micelles with different carrier to drug molar ratios was determined by hplc (table 2). The sizes of these ptx loaded peg - fts micelles were also evaluated under corresponding conditions . Peg5k - fts4 micelles could effectively solubilize ptx in aqueous solution at a molar ratio as low as 1:1 (m / m) with particle size around 50 nm (table 2). However, these ptx - loaded peg5k - fts4 micelles were only stable for 2.5 h. with an increase in carrier / drug ratio to 2.5/1, they formed mixed micelles with ptx that were stable for about 1 day . For the other three micelles, a minimal carrier / drug ratio of 2.5/1 was required to solubilize ptx . Overall, the conjugates with four molecules of fts worked better than the conjugates with two molecules of fts and peg5k conjugates were more effective than the peg2k counterparts in forming stable drug loaded micelles . The four conjugates were ranked as peg5k - fts4> peg2k - fts4> peg5k - fts2> peg2k- fts2 with respect to their efficiency in forming stable mixed micelles with ptx . Ptx concentration in micelles was kept at 1 mg / ml . Measured by dynamic light scattering particle sizer . Data mean that there was no noticeable size change during the follow - up period . The profile of ptx release from the four peg - fts micelles was examined by a dialysis method . As shown in figure 3, peg5k - fts4/ptx mixed micelles showed relatively slower kinetics of ptx release compared to other ptx - loaded peg - fts micelles . Cumulative ptx release profile from four ptx - loaded peg - fts micelles . Pbs containing 0.5% (w / v) tween 80 was used as the release medium . Ptx concentration was fixed at 0.5 mg ptx / ml . Values reported are the means sd for triplicate samples . As a delivery system for intravenous application, the potential detrimental interaction of figure 4 shows the hemolytic activities of drug - free peg2k - fts2, peg2k - fts4, peg5k - fts2, and peg5k - fts4 micelles . Polyethylenimine (pei), a cationic polymer known to have significant hemolytic effect, was included as a positive control . In contrast, all of the four drug - free peg - fts micelles displayed only negligible levels of hemolytic activities, suggesting that peg - fts micelles are mild surfactants suitable for in vivo delivery of hydrophobic anticancer drugs . In vitro hemolysis assay of four peg - fts micelles compared with pei . Rat rbcs were treated with the four different peg - fts micelles or pet at 0.2 and 1 mg / ml, respectively . The lysis of rbcs was determined by measuring the release of hemoglobin spectrophotometrically (= 540 nm). Rbcs incubated with pbs and triton x-100 (2%) were used as the negative and positive controls . The fts2 conjugates (peg2k - fts2 and peg5k - fts2) were more effective than fts4 conjugates (peg2k- fts4 and peg5k - fts4) in inhibiting the tumor cell proliferation . Similar results were found when the four conjugates were examined in hct-116 (figure 5b) and 4t1.2 cells (figure 5c). Cytotoxicity of four drug - free peg - fts micelles in mcf-7 human breast carcinoma cell line (a), hct-116 human colon carcinoma cell line (b), and 4t1.2 mouse breast cancer cell line (c). Cells were treated with different micelles for 72 h and cytotoxicity was determined by mtt assay . Figure 6 shows the protein expression level of total ras 20 h following treatment of hct-116 cells with the four different conjugates at a fts concentration of 40 m . However, the conjugates with two fts molecules were more active than the counterparts with four fts molecules in reducing the protein expression levels of ras in hct-116 cells . Effects of peg - fts micelles on total ras protein expression in hct-116 cells . Hct-116 cells were treated with four different peg - fts micelles for 20 h (at a fts concentration of 40 m). Figure 7 shows the cytotoxicity of free ptx (in dmso) and ptx formulated in peg2k - fts2, peg2k - fts4, peg5k - fts2 and peg5k - fts4 micelles in 4t1.2 cell line . Delivery of ptx via the four different peg - fts micelles led to varied levels of improvement . Nonetheless, ptx - loaded peg5k - fts4 micelles were more potent than ptx formulated in the other micellar formulations (peg2k - fts2, peg2k - fts4, and peg5k - fts2) in inhibiting the tumor cell growth . The ic50 of free ptx and the four micellar formulations of ptx are summarized in supporting information table s1 . Cytotoxicity of ptx - loaded peg - fts micelles in 4t1.2 mouse breast cancer cell line . Cells were treated with free ptx or different micellar formulations of ptx for 72 h and cytotoxicity was determined by mtt assay . Ptx - loaded peg5k - fts4 micelles showed an improvement in cell growth inhibition compared with other formulations . * p <0.05 (ptx / peg5k - fts4 vs ptx), p <0.05 (ptx / peg5k - fts4 vs ptx / peg5k - fts2 or ptx / peg2k - fts4), p <0.05 (ptx / peg5k - fts4 vs ptx / peg2k - fts2, ptx / peg5k - fts2, or ptx / peg2k - fts4). The in vivo therapeutic effectiveness of ptx formulated in peg2k - fts2, peg2k - fts4, peg5k - fts2, and peg5k - fts4 micelles was evaluated, respectively, in a syngeneic murine breast cancer model (4t1.2), and compared to taxol . As shown in figure 8a, ptx - loaded peg2k - fts2 micelles exhibited a similar tumor growth inhibitory effect compared to taxol treatment group . In contrast, ptx formulated in peg5k - fts2 and peg5k - fts4 micelles demonstrated a significantly enhanced antitumor activity compared to taxol (p <0.01). Furthermore, ptx formulated in peg5k - fts4 showed a trend of improvement in antitumor activity compared with ptx formulated in peg5k - fts2 (p = 0.09). No significant changes in body weight were noticed in all treatment groups compared to pbs control group (figure 8b). In addition, serum levels of transaminases in the mice with different treatments were comparable to those in pbs control group (table 3). Babl / c mice were inoculated s.c . With 4t1.2 cells (2 10 cells / mouse). Six days later, mice received various treatments twice a week and tumor growth was monitored and plotted as relative tumor volume (a). Significant improvement in antitumor activity was found for the ptx - loaded peg5k - fts4 group compared with the taxol group (* * p <0.01; n = 5) and the group of ptx - loaded peg2k - fts2 (* p <0.05; n = 5). Changes in body weights of mice in different treatment groups were also monitored (b). We have systematically compared the physicochemical property and the in vitro and in vivo ptx delivery efficiency of four peg - fts conjugates that vary in the length of peg motif (peg2k vs peg5k) and the molar ratio of peg / fts (1/2 vs 1/4). All of the four peg - fts micelles possessed very small sizes of 2030 nm . Doxil (doxorubicin hcl liposome) and abraxane (albumin - bound paclitaxel) are two fda - approved formulations . The particle sizes of doxil and abraxane are 150 and 130 nm, respectively . Their relatively large sizes may limit the diffusion in the tumor, and thus limit their therapeutic effectiveness . It has been reported that nanoparticles need to be smaller than 100 nm in order to circumvent macrophage clearance in the lungs and that particles of further reduced size (64 nm) are needed for effective penetration through neovasculatures to reach tumor cells . The small sizes of our peg - fts micelles shall ensure efficient passive targeting to the solid tumors . In addition to size, drug loading capacity and formulation stability are two other important features for an effective micellar system . Our data showed that the peg - fts conjugates with four fts molecules were significantly more effective than the conjugates with two molecules of fts in forming stable drug - loaded micelles . Previous studies have shown that increasing the ratio of hydrophobic / hydrophilic blocks is associated with increased drug loading capacity and enhanced formulation stability . The improved performance of peg - fts conjugates with an increased number of core forming units may be attributed to a similar mechanism . Fts, as a hydrophobic core of micelles, has a lipid chain and benzene ring structure . The lipid chain of fts contributes to the loading of hydrophobic drug through hydrophobic interaction . At the same time, the benzene ring of fts is capable of forming interaction with drug carrying aromatic ring structure . In the peg - fts micellar system, stacking and hydrophobic interaction are likely to work cooperatively to promote both drug / carrier and carrier / carrier interactions . Such interactions are also expected to be further enhanced with an increase in the number of fts molecules in peg - fts conjugates . A prominent advantage of peg modification is to impart the in vivo longevity to drug carriers . Torchilin s group has reported that peg / core ratio affected the performance of the micelles . Furthermore, the length of peg also acted on the cmc, which in turn influenced the performance of the micelles, particularly in vivo . Our data showed that peg5k - conjugates had a lower cmc and were more effective than peg2k - conjugates in forming stable micelles with ptx . One unique feature for peg - fts micellar delivery system is its intrinsic antitumor activity . The four different peg - fts conjugates showed varied levels of antitumor activity by themselves in three cancer cell lines . The conjugates with two fts molecules showed higher levels of cytotoxicity compared to the counterparts with four fts molecules . The higher levels of cytotoxicity for the conjugates with two fts molecules are unlikely due to a more active surface activity of the double chain conjugates as all of the four conjugates showed minimal hemolytic activity (figure 4). It is likely that active fts is more readily cleaved from the conjugates with two fts than the ones with four fts molecules due to less steric hindrance to intracellular esterases . We also compared the in vitro cytotoxicity of the four ptx - loaded peg - fts micelles (figure 7). Ptx - loaded peg5k - fts4 micelles showed better cytotoxicity than free ptx and ptx formulated in other three micelles in 4t1.2 cells . This is likely due to a more efficient intracellular delivery of ptx via peg5k - fts4 micelles because peg5k - fts4 formed the most stable micelles with ptx among the four micellar systems tested . In vivo therapy study clearly showed a significantly higher level of antitumor activity for ptx formulated in peg5k - fts4 micelles compared to either taxol or ptx formulated in peg2k - fts2 (figure 8a). This is likely due to the significantly improved stability for peg5k - fts4 micelles, which contributes to more effective delivery of ptx to tumor tissue in vivo . In summary, we have shown that peg5k - fts4 formed the most stable mixed micelles with ptx among four peg - fts micelles . Furthermore, ptx formulated in peg5k - fts4 micelles was more active in cytotoxicity than free ptx and ptx formulated in other three peg - fts micelles . In vivo, ptx - loaded peg5k - fts4 led to an improved tumor growth inhibitory effect in comparison to ptx formulated in peg2k - fts2, peg2k - fts4, and peg5k - fts2 as well as taxol in a syngeneic mouse model of breast cancer (4t1.2). More studies on the structure and activity relationship are needed to further improve the peg - fts - based delivery system.
Platinum drugs are a mainstay of cancer therapy . Approximately half of all cancer patients receiving chemotherapy cisplatin, carboplatin, and oxaliplatin (chart 1) three platinum complexes approved by the us fda for the treatment of human cancer are commonly applied to treat bladder, testicular, head and neck, ovarian, colon, and small cell and nonsmall cell lung cancers . Despite such widespread use, the cytotoxicity of these drugs is not limited to cancer cells, and off - target activity results in emesis, alopecia, nausea, kidney damage, myelosuppression, and peripheral neuropathy . Moreover, many tumors are either inherently resistant to the currently employed platinum - based therapies or acquire resistance during treatment . In an attempt to find molecules with improved potency, fewer side effects, and a novel spectrum of activity, researchers have prepared thousands of platinum complexes and tested them for anticancer activity . One strategy used to address the foregoing issues is to devise complexes that depart from the neutral, square - planar, dna - cross - linking cis - dia(m)mine a current manifestation explores cationic monofunctional platinum(ii) complexes, which bear only one labile ligand and form one bond to the dna nucleobases . The significant difference in the interaction of monofunctional complexes with dna compared to classical bifunctional cross - linking compounds very likely contributes to the unique response that phenanthriplatin, or cis-[pt(nh3)2(phenanthridine)cl] (chart 1), elicits when used to treat cancer cells . Studies with the monofunctional compound pyriplatin (chart 1), cis-[pt(nh3)2(pyridine)cl], reveal that little distortion of the dna double helix is induced upon platination, and a similar situation is likely to be obtained with phenanthriplatin . This result is very different from the significant dna bending at 1,2-intrastrand cross - links that occurs following treatment with bifunctional platinum agents such as cisplatin . Anonleaving group ligands are colored blue, and leaving group ligands are red . Nonleaving group ligands are colored blue, and leaving group ligands are red . Crystallographic and biochemical studies have revealed the mechanism by which pyriplatin exerts its anticancer activity, most likely transcription inhibition followed by consequent apoptosis . Structural studies suggest that the steric bulk of the pyridine ring is crucial for activity . This hypothesis provides an explanation for the contrast between the activity of pyriplatin and the inactivity previously observed for monofunctional compounds such as [pt(nh3)3cl] and [pt(dien)cl]. A systematic variation of the n - heterocyclic ring am in compounds of the form cis-[pt(nh3)2(am)cl] resulted in the discovery of the far more potent analog phenanthriplatin . In a preliminary screen of cultured human cancer cells, phenanthriplatin displayed significantly greater cytotoxicity than cisplatin and showed a pattern of activity distinct from that of either cisplatin / carboplatin or oxaliplatin . A more detailed understanding of the spectrum of activity was gained by analyzing the cytotoxicity of phenanthriplatin in the nci60 panel of cancer cells . The pattern of cell killing was uncorrelated with that of any other platinum agent in the nci database . Unlike pyriplatin, the asymmetry of the phenanthridine ring renders phenanthriplatin chiral . That phenanthriplatin can exist as two distinct enantiomers is of potential importance because the two enantiomers may display different pharmaceutical activity . Initial experiments revealed that phenanthriplatin binds dna and that it does so in a covalent, rather than intercalative, manner . Moreover, studies with e. coli, analogous to those initially performed to investigate the mechanism of action of cisplatin, corroborate the hypothesis that the interaction of phenanthriplatin with dna is responsible for its anticancer effects . To gain more insight into the nature of the interaction of phenanthriplatin with dna, we prepared small molecule complexes that model its reactions with guanosine residues (chart 2). The n7 position of guanine is the most nucleophilic among dna bases and, as a result, it is the primary binding site for platinum agents . The model complexes cis-[pt(nh3)2(r - gua)(am)](otf)2, where r - gua is a 9-alkylguanine, am is phenanthridine, and otf is trifluoromethanesulfonate or triflate, were therefore prepared using the triflate salt of phenanthriplatin as a synthetic precursor . The chirality of phenanthriplatin combined with coordination to r - gua creates diastereomers, the nature of which was investigated by x - ray crystallography and nmr spectroscopy . Here we report the results of an investigation of these diastereomeric analogs of phenanthriplatin (chart 2), which were prepared to investigate whether the two phenanthriplatin enantiomers can be resolved on the physiological time scale . Consideration of its dynamics is crucial for interpreting the conformational isomerism observed with the 9-alkylguanine model complexes . Evidence in both the solid state and solution phase indicates that, when phenanthriplatin reacts with guanine, diastereomeric selection occurs among the possible conformational isomers that can form . The origin of this selection has been identified, as described in this article . The synthesis and characterization of the compounds under discussion are presented in the supporting information along with crystallographic details and specifications of the instruments used for physical measurements . H nmr spectra were acquired over a temperature range for 24 to investigate potential fluxional behavior exhibited by these compounds . The rate of exchange at the temperature at which two peaks coalesce was estimated using eq 1, in which k is the rate of exchange at the coalescence temperature and is the difference in hz between the two signals in the low temperature (slow exchange) limit . This estimate was used to inform the initial guess in a full line shape analysis . Simulated spectra were fit to experimental spectra at the different temperatures, and the corresponding rate constants were extracted . These rate constants were used to construct eyring plots and determine activation parameters.1 deuterated dmso solutions of 6 and 8, both of which are 9-methylguanine complexes, were sparged with n2 for 2 min and then sealed in an nmr tube under a blanket of nitrogen . Briefly, for each sample, a h nmr spectrum was acquired following preirradiation at the frequency of the methylguanine h8 (h8 g) resonance . The preirradiation power was chosen so as to just saturate the h8 g signal and eliminate it from the spectrum . A second spectrum was then acquired with identical parameters, but with the saturation frequency set to a region downfield and devoid of signals . No cross peaks between h8 g and any of the phenanthridine protons were observed in a noesy experiment . Rotating - frame overhauser effect (roesy) spectra were collected on samples prepared in deoxygenated acetone - d6 . A mixing time of 200 ms was employed with a 90 pulse of 11 s . The complex cation of 6 was constructed in gaussview, and the planes of the phenanthridine and 9-ethylguanine ligands were set perpendicular to the coordination plane . Two conformers were investigated, one in which the h6 proton of the phenanthridine ligand (h6p) and h8 g were on the same side of the platinum coordination plane and the other in which they were on opposite sides . The zero order relativistic approximation (zora), along with the attendant tzv - zora basis set, was applied to treat relativistic effects . The resolution of the identity approximation and the appropriate auxiliary basis set were used to accelerate computations . The stationary nature of the structures obtained from geometry optimizations was confirmed using numerical frequency calculations . Optimizations were conducted in either the gas phase or in solution by using an implicit conductor - like screening model (cosmo). To model aqueous solvation, the dielectric constant of the polarizable continuum was set to 80.400 and the refractive index to 1.3300 . Monofunctional complexes having the formula cis-[pt(nh3)2(am)cl], where am is an n - heterocyclic ligand, have previously been obtained as nitrate salts by treating cisplatin, cis-[pt(nh3)2cl2], with 1 equiv of silver nitrate followed by an equivalent of am . Alternatively, cis-[pt(nh3)2(am)cl]cl can be obtained by heating cisplatin with am to displace one of the chloride ligands . A major problem with these methods, however, is that neither silver - mediated halide abstraction nor direct ligand substitution proceeds selectively at just one coordination site . As a result, in addition to the desired cis-[pt(nh3)2(am)cl] complex, appreciable amounts of cis-[pt(nh3)2(am)2] form together with unreacted cisplatin . Here we use silver triflate to prepare 7 and 1, triflate salts of pyriplatin and phenanthriplatin, respectively, to provide a much wider range and degree of solubility in organic solvents . As a result, addition of acetone to the residue that remains following removal of dmf from the synthesis mixtures of 1 and 7 dissolves the triflate salts of cis-[pt(nh3)2(am)cl] and cis-[pt(nh3)2(am)2]. Unreacted cisplatin, on the other hand, does not dissolve in acetone and can be removed by filtration . When ether is layered onto the acetone filtrate, crystals of cis-[pt(nh3)2(am)cl]otf deposit over the course of a few days . These crystals can be harvested before the more soluble cis-[pt(nh3)2(am)2]otf2 precipitates, providing access to analytically pure material . Spectroscopic characterization of these complexes was consistent with that which had been previously reported for the nitrate salts of these cations . The assignment of the peaks in the h nmr spectrum of 1 was carried out using a combination of cosy and noesy spectra (figure s4). The nmr spectra for these compounds (figure s19s22) show a multitude of peaks arising from different rotamers that interconvert slowly on the nmr time scale at room temperature . The syntheses of 5, 6, and 8 were achieved by treating either 1 or 7 with an additional equivalent of silver triflate and adding the appropriate 9-alkylguanine . As described above, the h nmr spectra of 24 exhibit peak multiplicity as a result of slow to intermediate exchange between conformational isomers . Upon heating, these signals broaden, coalesce, and finally sharpen as the rate of exchange increases . Although some regions of the spectra have complex overlapping features, other regions show well - resolved peaks and coalescence events . The line shapes of portions of the h nmr spectra of 24 that showed well - defined, baseline - resolved coalescing signals were simulated . The simulated spectra were fit to the experimental data by varying the rate constant . A two - site model was used for 2 and 4, and a four - site model was used for 3 . An example of the simulated and experimental data for 4 is shown in figure 1 . Data for all compounds can be found in the supporting information (figures s23s25). Experimental and simulated line shapes of portions of the h nmr spectrum of 4 . The temperature at which the experimental data were collected and the rate constant used to generate the simulation are shown next to each set of data . The variation in rate constant with temperature can be used to determine the activation parameters for the interconversion . The enthalpy and entropy of activation, h and s respectively, are obtained from the first order rate constants using an eyring analysis (figure 2). Eyring plots for all the coalescence events simulated are presented in the supporting information . The gibbs free energy of activation at a given temperature t, gt, can be obtained using eq 2.23the values of g298.15 for compounds 24 are collected in table 1 . Using eq 3, where kb is boltzmann s constant, h is planck s constant, and r is the universal gas constant, the rate constant at a given temperature, the lifetime of the molecule in a given conformational state at temperature t, t, for the process can be obtained by taking the inverse of the rate constant . The errors for g298.15 are obtained by standard propagation of the errors of h and s obtained from the least - squares linear regression eyring plots . The errors should be taken only as estimates of the true errors and are presented to indicate the relative precision with which the different determinations were made . The approximate nature of the error estimate arises from the fact that this analysis treats data across a logarithmic scale on equal footing . Moreover, it equally weights the rate constants from near the coalescence point, which are more accurate, and rate constants obtained far from coalescence, which are less accurate . The error propagation was not carried out for the lifetimes because only modification by physical constants is involved . Eyring plot of the conformational isomerism of 4 using the data from figure 1 . Pertinent crystallographic data for 1, 5, 7, and 8 are summarized in table s1 . The crystal structure of 1 (figure 3) shows the cis-[pt(nh3)2(phenanthridine)cl] cation to be similar in structure to that present in the structure of the nitrate salt, with bond lengths and angles falling within expected ranges . The geometry of the primary coordination sphere in both structures is essentially identical with an rmsd of 0.023 . The most significant differences are found in the orientation of the phenanthridine rings as shown in figure s30 . In the nitrate salt, the phenanthridine plane does not contain the line connecting the platinum atom and the nitrogen atoms of the phenanthridine and the trans ammine . This line instead forms an angle of 18 with the phenanthridine plane . In the triflate salt, this angle is only 10. an important aspect of the structure of the complex is that the asymmetry of the phenanthridine ligand about the platinum coordination plane produces a chiral molecule . Molecular diagrams of the platinum complexes from the crystal structures of 1, 5, 7, and 8 with thermal ellipsoids drawn at the 50% probability level . Color code: n blue, o red, c gray, cl green, pt magenta, h open circles . The chirality originates about the bond between the platinum center and the phenanthridine nitrogen atom (pt np) and can be classified according to the conventions of axial chirality . Np vector, the platinum coordination plane lies in front of the perpendicular plane of the phenanthridine ring . The direction in which the front ring needs to be rotated so as to have the priority substituent of the front plane coincide with the priority substituent of the back plane dictates the stereochemistry . Clockwise rotation is denoted p and counterclockwise rotation m. the different enantiomers of phenanthriplatin are shown in figure 4 . (a) the enantiomers of phenanthriplatin and (b) the convention used to classify them . In part b, the complex is viewed along the ammine platinum phenanthridine vector . The coordination plane is shown as a darkened line, and the phenanthridine plane as a dashed line . The structure of 7 (figure 3), the triflate salt of pyriplatin, also displays expected bond lengths and angles . In this complex, unlike 1, the line connecting the platinum atom and the nitrogen atoms of the pyridine and the trans ammine is essentially contained by the plane of the n - heterocycle . The ring deviates significantly, however, from a perpendicular orientation with respect to the platinum coordination plane . The dihedral angle of 60 between the pyridine and the coordination plane is consistent with the angle of 56 observed for trans-[pt(pyridine)2cl2]. Details of the determination of the structure of a nonmerohedral trilling of this latter compound are presented in the supporting information . Given the lack of steric or electronic factors to enforce strict perpendicularity between the pyridine ring and the coordination plane, the angle in both cases is most likely dictated by crystal packing interactions . The solution and refinement of the structure of 8 (figure 3) proceeded smoothly, except for the presence of a void about the 0, 0, 1/4 special position containing disordered electron density . The density could not be successfully modeled as either a molecule of dmf, diethyl ether, or a 1:1 disorder of the two, any of which would be consistent with the 41 e within the void . One dmf molecule disordered across each of the 4 voids of 228 within the unit cell (z = 8) would be consistent with the combustion analysis results obtained from this material . The pyridine and 9-methylgunanine rings are both canted in the same direction by 19 and 23, respectively . The structure of 5, the product of the reaction of activated 1 with 9-ethylguanine, was also solved (figure 3). The salt crystallized along with one water molecule, located on the 2-fold proper rotation axis, and one disordered acetone molecule . The presence of 0.5 equiv of water is consistent with the elemental analysis of this compound . The acetone present in the structure is not observed during combustion analysis and is probably removed during the vacuum drying of the substance . The phenanthridine ligands lie parallel to the ac plane, and the 9-ethylguanine ligands and the platinum coordination plane lie perpendicular to this crystallographic plane . The phenanthridine and 9-ethylguanine ligands of the square - planar complex cation the former is oriented essentially perpendicular to the coordination plane but the latter is canted by 23, such that the guanine carbonyl oxygen approaches the ammine coordinated cis to it . Preirradiation at the frequency of the h8 g signal in solutions of 6 and 8 induced perturbations in the signals of those protons that interact with h8 g in a through - space manner . In the difference spectrum of 8, obtained as described above, negative peaks were seen arising from the ch3 protons of the 9-methyl group as well as the ortho hydrogen atoms of the pyridine ring (figure s26). In the difference spectrum of 6, a negative peak was again seen for the ch3 protons of the 9-methyl group . Negative peaks were also observed for h6 and h7 of the phenanthridine ring (figure s27) owing to their close proximity to h8 g . The roesy spectrum of 6 also confirms the presence of the through - space interaction between h8 g and h6 of the phenanthridine ring (figure s28). Geometry optimizations were performed on two conformational isomers of 5 that are related by a 180 rotation about the pt np bond . In calculations in which the starting geometry had both n - heterocyclic ligands perpendicular to the coordination plane, optimization did not significantly alter the geometries of either of the conformers, which exhibited a negligible difference in strain energy . An overlay of the optimized structures of both conformers is shown in figure 5 . Overlay of the molecular mechanics optimized geometries obtained by setting the aromatic ligands perpendicular to the platinum coordination plane and rotating 180 about the platinum more rigorous geometry optimization using dft methods reproduced the canting of the alkylguanine ligand that was observed in the crystal structure of 5 (figure 6). In the gas phase, the distance of 2.72 between the carbonyl oxygen and the ammine nitrogen is sufficiently small to propose the presence of an intramolecular hydrogen bond . The calculations were also carried out with implicit aqueous solvation . As expected, the interaction between the carbonyl oxygen atom and the ammine nitrogen atom is attenuated, but the resulting on distance of 2.86 suggests that the interaction persists even in the presence of highly polar solvents . The canting of the guanine ring in (a) the crystal structure and (b) the dft - optimized structure of 5 . Color code: n blue, o red, c gray, pt magenta, h atoms omitted for clarity . (c) schematic representation of the dihedral angle formed between the platinum coordination plane (black) and the phenanthridine ring of 5 in the mm calculations (red), the crystal structure (blue), the dft calculations with implicit water solvation (purple), and the dft calculations in the gas phase (green). Studies with pyriplatin showed that this compound has a spectrum of activity that differs significantly from those of the clinically employed platinum anticancer drugs . The low potency of pyriplatin prompted a search for molecules that maintain this distinct spectrum of activity, but display higher activity . Phenanthriplatin was developed as the result of a systematic variation of the n - heterocyclic amine ligand, am, of cis-[pt(nh3)2(am)cl]. It was found to be 740-fold more potent that cisplatin across a variety of cell lines, and the distinct spectrum of activity was maintained . In an effort to better understand the mechanism of anticancer action of phenanthriplatin, we sought to investigate the structures of the adducts formed by phenanthriplatin and analogs of guanosine . For these studies, the triflate salt of the cis-[pt(nh3)2(am)cl] cation was prepared to facilitate subsequent synthetic steps . In the process of analyzing the solid state structure of the compound, however, we realized an aspect of the structure of this complex that had previously gone unnoticed: in the solid state, the complex cation is chiral . The centrosymmetric space group of the structure requires that both enantiomers be present in the crystal in equal portions . The chirality arises from the asymmetry of the phenanthridine ring about the coordination plane to which it is perpendicular . Different enantiomers of pharmaceutics typically display different activity because these molecules interact with biological systems that are inherently chiral . One example of direct relevance to the field of platinum anticancer research is oxaliplatin, which contains only one enantiomer in the form that is marketed for clinical use . The r, r isomer of trans - diaminocyclohexaneoxalatoplatinum(ii) has greater activity than that of the enantiomeric s, s form . If phenanthriplatin is indeed a racemic mixture of two stable enantiomers, then one might have significantly different activity than the other . It is crucial to realize, however, that the two enantiomers can interconvert via rotation about pt np . If rotation about this bond is rapid at ambient or physiological temperatures, then the complex is effectively achiral . As will be discussed below, in addition to implications for the enantiomeric resolution of the compound, asymmetry of the phenanthridine ligand about the platinum coordination plane also has implications for its interaction with dna . Investigation of the phenanthridine rotation is similar in nature to studies that have been carried out on models and retro - models of 1,2-d(gpg) intrastrand cross - links formed by bifunctional platinum compounds . This similarity arises from the fact, as will become important below, that both the phenanthridine ligand and guanine derivatives coordinate in a manner that is asymmetric about the platinum coordination plane . The chirality present in models of the intrastrand [pt(nh3)2{d(gpg)}] adduct was first recognized by cramer et al . The phenomenon has subsequently been investigated in great detail by marzilli, natile, and co - workers, among others . Dynamic nmr spectroscopy is a method ideally suited to investigate whether rotation about a chemical bond is hindered . Np in phenanthriplatin interconverts two enantiomers with identical nmr properties, however, and so this dynamic process will induce no change in the line shape of the spectrum, regardless of the rate at which it occurs . Accordingly, the room temperature h nmr spectrum of 1 shows a single set of well - defined resonances . The signals in the aromatic region are particularly sharp, and the breadth of the signals arising from the two ammines is due to a combination of quadrupolar relaxation from n and coupling to csa - broadened pt . To investigate rotation about the pt np bond, the rotamers must be rendered diastereomeric . This goal was accomplished by replacing the two ammine ligands with the enantiomerically pure r, r - diaminocyclohexane (dach) chelate . The c2 symmetry of this ligand ensures that the coordination sites trans to the two coordinated nitrogen atoms are chemically equivalent . In the room temperature h nmr spectrum of 2, the signals arising from the protons on the aromatic ring are all cleanly doubled (figures 1 and s19). Moreover, raising the temperature of the sample induces broadening, coalescence, and subsequent sharpening of the signals (figure s19). This behavior is consistent with the presence of two rotamers that interconvert rapidly on the nmr time scale at elevated temperature . The conformations of these two rotamers are equivalent to those of the enantiomers of phenanthriplatin, i.e. M and p, giving rise to (r, r)m and (r, r)p diastereomers . Detailed information about the energetics of the rotamer interconversion can be obtained by simulating the experimental line shapes obtained at different temperatures . The fit of the simulation to the experimental data can be optimized by varying k, the rate constant for interconversion . These k values obtained at different temperatures can be used to construct an eyring plot from which the activation parameters for the interconversion can be extracted . The gibbs free energy of activation for the interconversion of the (r, r)m and (r, r)p diastereomers of 2 at room temperature, g298.15, was 70 kj mol, and the corresponding rate constant at this temperature was 3.2 s. the inverse of this rate constant reveals that 63% of a diastereomerically pure sample of 2 would racemize within about 300 ms of dissolution at ambient conditions . The validity of this conclusion, however, rests on the accuracy with which 2 models the structure of 1 . The most significant difference of relevance to the discussion at hand is in the n n angle formed by either the ammines of 1 or the dach of 2 . The former, obtained from the crystal structure of 1, is 89.1, and the latter, from the crystal structure of oxaliplatin, is 83.5. the smaller angle enforced by the chelator relieves the steric interactions that provide a barrier to rotation of the phenanthridine ring . The barrier obtained with 2, therefore, provides an upper estimate to the rate of rotation in phenanthriplatin . Np in phenanthriplatin will be sufficiently slower that enantiomeric resolution may be possible . To obtain a diastereomeric analog of 1 in which the n pt n angle formed by the ammines is left unperturbed, 4 was prepared . The addition of a second pt np bond as a center of chirality creates (p, m), (m, p), (p, p), and (m, m) conformational isomers . The (p, p) and (m, m) designations describe a meso compound, and the (p, m) and (m, p) rotamers are enantiomers . Hindered rotation about the pt np bonds would, therefore, give rise to two sets of signals . Using a dynamic nmr analysis analogous to that described above for 2, activation parameters can be extracted (table 1, figure s25). In addition to adding a second chiral center, however, the second phenanthridine ligand may introduce steric bulk that inhibits pt np rotation to a degree greater than in 1 . To assess the influence of the cis disposition of two phenanthridine rings on pt np rotation, 3 was prepared . The set of conformers listed for 3 is also present for 4, but the presence of the r, r - dach ligand renders all four rotamers diastereomeric: (r, r)(p, m), (r, r)(m, p), (r, r)(p, p), and (r, r)(m, m). Dynamic nmr spectroscopy again reveals the activation parameters for interconversion (table 1) through a simulation of the line shapes of the four distinct sets of h nmr signals that coalesce on heating (figure s24). A comparison of the results obtained from the dynamic nmr experiments on 24 is shown in scheme 1 . Upon transitioning from 2 to 3, the addition of the extra phenanthridine ligand raised the barrier to rotation, but the lifetime only changed by a factor of 1.7 . Exchanging the chelating dach present in 3 for the two ammine ligands in 4 increased the lifetime by an order of magnitude . Even so, the lifetime of a given diastereomer of 4 is about 5 s. importantly, unlike 2, 4 provides an upper estimate of the barrier to rotation in phenanthriplatin . The results with 3 and 2 indicate that transitioning from 4 to 1 will lower the barrier to phenanthridine rotation . Np bond in phenanthriplatin is rapid on the pharmacological time scale and that enhanced activity cannot be obtained by isolating and administering one of the isolated enantiomers . The rotation about this bond does, however, play a significant role in the interaction of 1 with dna . The interaction of phenanthriplatin with dna can be modeled by preparing complexes with the formula cis-[pt(nh3)2(phenanthridine)(9-alkylguanine)]. Compounds 6 and 5 (chart 2) were prepared using 9-methyl- and 9-ethylguanine, respectively . This mode of coordination is corroborated by the shift in the h8gh nmr signal observed on binding to the platinum . As a result of this coordination geometry, the bond between the platinum center and the n7 of the guanine (pt ng) acts as a center of chirality in the same manner as pt np . Multiplicity in the h signals was expected to arise due to the slow interconversion of (pp, mg), (mp, pg), (pp, pg), and (mp, mg), where the subscripts indicate the coordinate bond to either phenanthridine (p) or 9-alkylguanine (g). Surprisingly, however, only a single set of signals was observed (figure s8). Cooling an nmr sample to 60 c did not induce any decoalescence or broadening (figure s22). In these molecules, therefore, either intramolecular rotation is occurring significantly more rapidly than in complexes 14 or one particular set of conformers is preferentially formed . Note that, for instance, (pp, pg) and (mp, mg) are enantiomers and would produce identical nmr spectra . Through - space dipolar interactions between nmr active nuclei provide an ideal means with which to probe three - dimensional molecular structure in solution . Before investigating the more complex phenanthridine - containing compounds, however, a simpler pyriplatin analog was prepared and studied . In 8, the pyridine ring is symmetric about the platinum coordination plane and so the platinum pyridine bond is not a center of chirality . The chirality about pt ng produces enantiomers but not diastereomers . In the crystal structure of 8, there are 2 sets of protons that are close enough to suggest that a nuclear overhauser effect (noe) will occur involving h8 g: the ch3 of the 9-methyl group (2.52) and the ortho protons from the pyridine ring (3.56). The methyl group provides a particularly convenient internal standard because the rigidity of the guanine ring ensures that the methyl protons will stay in close proximity to h8 g regardless of the relative orientation of the pyridine and guanine rings . A h saturation transfer nmr experiment was carried out with preirradiation at the frequency of the h8 g signal and revealed a through - space interaction between h8 g and the 9-methyl protons as well as the ortho protons of the pyridine ring (figure s26). In the (mp, pg) isomer of 6, as well as the enantiomeric (pp, mg), h8 g and h6p are on the same side of the coordination plane . In (pp, pg) and (mp, mg), which are diastereomers of the first pair, h8 g and h4p are on the same side of the platinum coordination plane . Only those protons that are on the same side of the platinum coordination plane as h8 g are expected to undergo through - space interactions with this nucleus . When a saturation transfer experiment analogous to that described for 8 was conducted with 6, an interaction was observed between h8 g and the 9-methyl protons, as expected, and with the h6p proton . Moreover, an interaction was also seen with h7p, further confirming that the (mp, pg)/(pp, mg) enantiomeric pair is preferentially formed in solution over the (pp, pg)/(mp, mg) pair . A though - space interaction was also observed in the roesy spectrum (figure s28). A crystal structure of 5 was solved (figure 3), and it also showed only the presence of (mp, pg) and (pp, mg). The centrosymmetry of the space group c2/c in which the complex crystallized requires the presence of both enantiomers . Molecular mechanics calculations were initially performed to interrogate the origin of the diastereoselectivity exhibited by 5 and 6 . Calculations in which the aromatic ligands were set perpendicular to the coordination plane (figure 5) revealed little energetic difference between the two diastereomeric forms . Inspection of the crystal structure of 5, however, reveals that the guanine ligand is not perpendicular to the coordination plane . Rather, it cants so as to direct the carbonyl oxygen toward the cis - coordinated ammine . The on distance of 3.19 is too long to constitute a formal intramolecular hydrogen bond in the solid state, but such an interaction may occur in solution . A similar interaction between the carbonyl of a pyriplatin - platinated guanosine and the cis ammine of the platinum complex was observed in the crystal structure of pyriplatin - platinated dodecamer duplex dna . Dft geometry optimization was able to reproduce the canting of the 9-alkylguanine . In the optimized gas phase geometry, inclusion of implicit aqueous solvation weakens the interaction somewhat, but the on separation remains short at 2.86 . The origin of the diastereoselectivity appears to result from this canting of the guanine ligand, which relieves steric congestion over one face of the platinum complex . One side of the phenanthridine ligand has a greater degree of steric bulk in the vicinity of the platinum center than the other, as illustrated in figure 6 . In the favored diastereomer, this bulkier portion of the phenanthridine is directed toward the vacant space formed by the canting of the guanine . It remains to be seen whether the conformational preference is maintained in full length dna polymers or during complexation with proteins that recognize platinum lesions . As a final comment n bonds in phenanthriplatin were not rapid, then different diastereomeric forms of the platinum adduct could be kinetically trapped regardless of the energetic preference that might exist for one conformer . Instead, the unhindered bond rotation established above allows the complex to assume the thermodynamically favorable conformation, regardless of the manner in which it initially binds to the 9-alkylguanine . The solid state structure of phenanthriplatin highlights the fact that asymmetry of the phenanthridine ligand about the platinum coordination plane results in chirality . The use of model compounds with diastereomeric rotamers provides evidence that rotation about this bond in phenanthriplatin is rapid, eliminating any need to isolate and administer an enantiomerically pure compound . Rapid rotation about the this bond also prevents the kinetic resolution of diastereomers in complexes of the type cis-[pt(nh3)2(phenanthridine)(9-alkylguanine)], which mimic the interaction of phenanthriplatin with dna . The (mp, pg) and (pp, mg) diastereomers observed in both solution and the solid state are those in which the h8 g and h6p are located on the same side of the platinum coordination plane . The experimental data described above indicate that interaction between the guanine carbonyl and cis - coordinated ammine determines the preferential diastereomer formation.
The ministry of health, labour and welfare of japan has recommended shortening of the length of hospital stays1 . In general, shortening the length of stay contributes to a reduction in the physical and mental stress of patients and rapidly returns them to their jobs and communities . Actually, in a previous study, it was shown that a shorter length of stay resulted in not only a financial benefit but also a patient benefit2 . The patient s benefit is spending less time out of their home and reduction of the possibility of contracting nosocomial infections during a stay in the hospital2 . In stroke patients, stroke - related impairment, medical complications, family support, and discharge destination predict the length of stay3 . In another previous study, impairment, activities of daily living (adl), unplanned discharges, and discharge to facilities affected the length of stay4 . On the other hand, similarly, patient - related factors were found to affect rehabilitation outcome, whereas illness- and intervention - related factors did not6 . This behavior pattern includes impatience, urgency, aggressiveness, and particularity about details . The type a behavior pattern is regarded as an independent risk factor of cardiovascular diseases7 . However, patients with type a behavior pattern characteristics may have better compliance with rehabilitation and other forms of medical and social support than those with non - type a behavior . If patients with the type a behavior pattern have better compliance, they would have a shorter length of hospital stay than those with non - type a behavior . To examine this hypothesis, in this study, we compared the length of stay of patients with the type a behavior pattern with that of patients with a non - type a behavior pattern . We hypothesized that length of stay of patients with the type a behavior pattern would be shorter than that of patients with a non - type a behavior pattern . Fifty - seven patients participated in this study . All patients had stayed in the comprehensive rehabilitation unit of omaezaki municipal hospital between april and december 2013 . The exclusion criteria were as follows: transfer to another hospital, medical facility, or welfare facility during the research term and refusal to provide informed consent . Type a behavior pattern was assessed by an abbreviated set of 12 questions developed by maeda8 . Hardly were scored as 2, 1, and 0 points, respectively, for nine questions, and the points were doubled for three questions . A total score of 17 or greater was defined as type a, and the other subjects were defined as non - type a. after the subjects were discharged, we counted the number of days between admission and discharge to evaluate the length of stay . To evaluate the ability to perform adl at discharge, we measured barthel index for all patients . We used the student s t - test to examine statistical differences in length of stay and barthel index at discharge between subjects with type a behavior and those without type a behavior . We analyzed these measurements by statistical package for social sciences (spss) version 19 . Ultimately, 55 subjects (75.311.7 years old, 18 males and 37 females) were classified into either the type a group (n = 26) or the non - type a group (n = 29), as shown in table 1table 1.characteristics of the type a and non - type a behavior patternscharacteristicstype a groupnon - type a grouppatients (n)2629male (n)108female (n)1621cause of admissionstroke (n)810fractures (n)1310disuse syndrome (n)23others (n)36values represent number of subjects . Others causes of admission include spinal code injury, spinal canal stenosis, and total knee arthroplasty .. the characteristics of subjects (sex and cause of admission) were similar in the two groups . Age was not significantly different between the two groups . Also, the barthel index at discharge was not significantly different between the two groups . However, length of stay was significantly higher in the non - type a group compared with the type a group (p <0.05) (table 2table 2.difference in length of stay between the type a group and non - type a groupscharacteristicstype a groupnon - type a groupage (years)77.271.4discharge bi97.5897.08length of stay (days)66.287.4 * * p <0.05 vs. type a group, values represent averages . Others causes of admission include spinal code injury, spinal canal stenosis, and total knee arthroplasty . Our results support the hypothesis that patients with the type a behavior pattern have better compliance with forms of medical support than patients with a non - type a behavior pattern . Patients with the type a behavior pattern had a shorter length of hospital stay than patients with a non - type a behavior pattern . Length of hospital stay can be affected by multiple factors, including medical complication, family support, discharge destination3, impairment, adl4, and rehabilitation9 . Patient - related factors5, especially psychological factors6, affect rehabilitation outcomes . The subjects in this study were patients who stayed in a comprehensive rehabilitation unit . Patients who stay in a comprehensive rehabilitation unit do not require much medical care and are able to focus on rehabilitation . Hence, our data suggest that psychological factors of the patients, such as type a behavior, affect rehabilitation compliance . Length of stay, ability to perform adl at discharge, and patient age influence rehabilitation outcome and whether patients can return home or not10, 11 . There was no significant difference in barthel index or patient age between patients with the type a behavior pattern and those with a non - type a behavior pattern . Thus, it appears likely that ability to perform adl at discharge, patient age, and destination at discharge do not influence length of stay . The type a behavior pattern includes impatience, urgency, aggressiveness, and particularity about details . Patients who have higher levels of physical activity during rehabilitation were associated with a shorter length of stay12 . Because patients with the type a behavior pattern might have good compliance with rehabilitation and increase their physical activity the type a behavior pattern is well known to be an independent risk factor of cardiovascular diseases7 . Also in the japanese, the type a behavior pattern is associated with an increased risk of acute myocardial infarction, especially in women13 . So, the type a behavior pattern may be considered not a good characteristics in general . In contrast, another article reported that the type a behavior pattern reduced the risk of coronary heart diseases in japanese men14 . Japanese men who do not express their anger may have an increased risk of high blood pressure15 . This previous article indicates that non - type a japanese men may have a higher hypertension risk than type a japanese men . In the japanese, the type our results also show that patients with the type a behavior pattern may be able to return home early . Finally, the cause of a short length of stay may not be rehabilitation compliance . In conclusion, our results suggest that the type a behavior pattern shortens the length of hospital stay . The data show that we should consider the patient s characteristics in rehabilitation to protect the patient and for financial benefit.
Peritoneal carcinomatosis (pc) is associated with a poor prognosis, and, once it is diagnosed, survival is generally less than 6 months [1, 2]. Peritoneal carcinomatosis represents a devastating form of cancer progression and the pathogenesis of this clinical entity can be explained by several biological models and a better understanding of underlying tumor kinetics and cellular dissemination mechanisms . A considerable number of patients presenting with peritoneal carcinomatosis from digestive or gynecological cancers are aged 70 or older . Offering a safe and appropriate treatment to elderly patients with pc presents a challenge for healthcare resources . The combination of cytoreductive surgery (crs) and hyperthermic intraperitoneal chemotherapy (hipec) plays an important therapeutic role in patients with pc . On the other hand, elderly patients are traditionally associated more frequently with comorbidities and a reduced capacity to recover or tolerate aggressive surgery [5, 6]. Several recent results have shown that age alone does not influence the outcome of surgery and cancer - specific survival in these patients is similar to that of younger patients [79]. As for the management of pc nowadays, many patients are treated in specialized centers around the world with extensive crs with peritonectomy procedures combined with hipec and encouraging disease - free and overall survival results have been reported . In the past, in the few randomized trials that were performed, age was used as a selection criterion, investigating the outcomes in patients younger than 70 or 65 years [10, 11]. However, patients with peritoneal carcinomatosis stemming from any tumor site are 70 years old or older at the time of diagnosis . The risks and benefits of crs and hipec in elderly patients using prospectively collected data from our institution with specialized interest in peritoneal surface malignancies, we examined the outcomes in the elderly patients who underwent crs and hipec . From a pool of 100 patients with a diagnosis of pc who underwent crs and hipec in our center in greece, in this study we have included patients at an age of 70 years or older and the results were compared to the patients younger than 70 years . Data regarding patient characteristics, surgical procedures, perioperative outcomes, and survival outcomes were prospectively collected . Regular followup was performed every three months for the first year and at six - month interval thereafter . The eligibility for crs and hipec procedures is decided after physical examination and double contrast - enhanced ct scans of the chest, abdomen, and pelvis . In addition, a positron emission tomography (pet) scan was performed to assess the extent of disease if necessary . Each case was put to discussion at a multidisciplinary team meeting attended by surgical oncologists, medical oncologists, radiation oncologists, anaesthesiologists, cancer care nurses, and research staff . In all patients a jugular or subclavian central venous catheter was placed and they received prophylactic antibiotic treatment with cefazolin 200 mg and metronidazole 500 mg every six hours during surgery and postoperatively . Procedures were performed by a specialized surgical team, led by the same surgeon (js). Patient position was supine; a midline laparotomy was performed, followed by assessment of the extent of disease with the use of the peritoneal cancer index (pci). The pci is a combined assessment of thickness of the lesion, size and distribution of tumor deposits in different abdominal regions, resulting in a numerical score which represents a quantification of the extent of the disease . The macroscopic result of cytoreduction was assessed and recorded using the completeness of cytoreduction (cc) score . After cytoreduction, hipec was performed by infusion of a heated chemoperfusate into the abdomen using either the coliseum technique or the closed abdomen technique at approximately 42.5c for 90 minutes . The drug protocol that was used has been described previously by our team, with a 30% dose reduction in the aged group . Overall survival was defined as the time between the crs and hipec and the date of death or last followup . Disease - free survival was defined as the time between the crs and hipec and the date of recurrence thirty patients of 70 years or older (mean age 74.5 years) underwent combined treatment with crs and hipec . Details on concomitant disease and medical history are given in table 1 . In all patients, median time between the primary tumor resection and crs + hipec was 18 months (6180). In the older group, the mean pci was 25 (439) and a complete removal of the peritoneal disease (ccs0) was achieved in 16 patients (53.3%), while in the younger group (<70 years) the mean pci was 24 (339) and a ccs0 was achieved in 55.7% . All the data concerning the intraperitoneal procedures are presented in table 2 . In fifteen patients (50%) of the aged group, the crs and hipec were uncomplicated . In the group under 70 years, in 41 patients (58.5%) no complications were observed . The mortality rate was 1/30 (3.3%) in the older group versus 1/70 (1.4%) in the younger group, respectively (ns). Three patients from the older group (10%) required surgical intervention in order to deal with acute and severe complications compared to five patients (7.1%) from the younger group . Age plays a role in the overall survival rates three years after the initial operation . Univariate analysis was performed to assess the influence on overall survival of the following factors: age> 75, histologic type of tumor (mucinous adenocarcinoma versus adenocarcinoma), peritoneal cancer index (pci), completeness of cytoreduction score (ccs), amount of blood transfused, duration of crs + hipec, and occurrence of postoperative complications . Low pci (<10), completeness of cytoreduction (ccs0), and tumor histology were the factors that influenced overall survival . Over the past 30 years the global burden of cancer has more than doubled, so the ageing population and the associated rise in cancer prevalence lead to an increase in demand of cancer treatment . These factors together with the availability of modern surgical equipment, new antibiotic regimens, and the high standards of anesthetic and icu care may reduce the risk of age as a preoperative selection criterion . Studies from other centers confirm that crs + hipec can be safely performed in elderly patients, including octogenarians [17, 18]. In the past, age has been regarded as a limiting factor for aggressive or curative therapies . In the last decade, reports have shown acceptable outcomes in terms of morbidity and mortality in patients older than 70 years of age undergoing gastrectomy for gastric cancer, pancreatectomy for pancreatic cancer, or liver metastasectomies . Our study shows that crs + hipec can be performed safely in patients aged 70 years or older with an acceptable morbidity and mortality, comparable to patients younger than 70 years . Primary outcomes in our study were overall survival, which is 52% in five years for the younger group versus the older group, in which it is 30% . This result demonstrates that three years after the initial operation there is a survival benefit in the younger population . To optimize the outcomes of elderly patients undergoing crs + hipec low pci (<10), completeness of cytoreduction (ccs0), and tumor histology were the factors that influenced overall survival . Demonstrates equal results concerning the morbidity and mortality in elderly colorectal patients undergoing crs + hipec . The study presents the results in twenty - four patients aged> 70 years, but they did not compare their results against adults <70 years . Also foster et al . Presented a retrospective analysis of twenty patients over the age of 65 who presented for consideration of crs + hipec and suggested that crs + hipec can be safely performed in elderly patients including octogenarians . Other studies in the past have evaluated the safety, effectiveness, and feasibility of other major abdominal operations in the elderly, such as liver, pancreatic, gastric, and ovarian cancer surgery, showing a low mortality and acceptable morbidity with a careful patient selection process [1922]. The role of hipec in the management of peritoneal carcinomatosis worldwide remains unclear with excellent result in well selected patients and especially in those of colorectal and ovarian origin . Over the past ten years, randomized controlled trials have shown prolongation of survival with the combination treatment as compared with standard care of conventional surgery and systemic chemotherapy . The studies in elderly population have a bias concerning the quality of life and the influence of this aggressive approach of treatment in the comorbidities and its role in long - term survival . This factor may play a role in the survival of aged patients in our study, who, after a three - year period, have statistically significant lower survival rates compared to the younger group . Votanopoulos et al . Recently reported a series of 81 patients older than 70 years who underwent cytoreductive surgery and hipec . The 30-day mortality was 13.8% and severe postoperative complications occurred in 38% of the patients, results which are similar to ours . In conclusion, more data are needed to assess the survival rates and to identify factors of influence on morbidity, mortality, and quality of life of patients receiving crs + hipec . Also the treatment is feasible in patients over 70 years with good performance status and is well selected after consideration of the tumor volume, grading, and the type of resection . These factors must be the inclusion criteria for elderly patients in clinical trials to determine the best results.
Briefly, poly(-caprolactone) (pcl) were dissolved at 143 mg / ml in equal parts tetrahydrofuran and n, n - dimethylformamide, then extruded at 2.5 ml / h through a spinneret charged to + 13 kv . The resulting nanofibrous jet was collected on a grounded mandrel rotating at 10 m / s and located 20 cm from the spinneret . Aluminum shields on either side of the spinneret were charged to + 9 kv to focus the jet . The spinneret was fanned back and forth to ensure uniform fiber deposition . Mscs were isolated from femoral and tibial bone marrow of 3 - 6 month old calves and expanded to passage 2 as described previously . Scaffolds were hydrated by sequential washes in 100%, 70%, 50% and 30% ethanol and finally phosphate buffered saline (pbs). Before seeding, 50 l of cell solution (1 10 cells / ml) were applied to one side, followed by incubation at 37c for one hour . Scaffolds were then turned and an additional 50 l of cell solution applied to the other side . After two hours further incubation, samples were transferred to chemically defined media (dmem, 0.1m dexamethasone, 40 g / ml lproline, 100 g / ml sodium pyruvate, 1% insulin, transferrin, selenium / premix, and 1% penicillin, streptomycin and fungizone supplemented with 10 ng / ml transforming growth factor 3). Media was replaced twice weekly for the duration of the study . After measuring cross - sectional area using a custom laser device, samples (n=5) were clamped with serrated grips and loaded into an instron 5542 testing device . All testing was performed in a pbs bath . The mechanical testing protocol consisted of: (1) a nominal tare load of 0.1n applied at 0.1% strain / sec, followed by stress relaxation for 5 minutes, (2) 15 preconditioning cycles to 0.1% strain at 0.05% strain / sec, and (3) a quasi - static elongation at 0.1% strain / sec until failure . Strain was determined as extension normalized to gauge length; stress was computed as the load normalized to initial cross - sectional area . Modulus was computed as the slope of the stress - strain plot, determined by regression to the linear portion of the curve . For biochemical analyses, samples were digested for 16 hours in papain at 60c, then analyzed for s - gag content using the 1,9-dimethylmethylene blue dye - binding assay, for orthohydroxyproline (ohp) content (after acid hydrolysis) to determine collagen content by reaction with chloramine t and dimethylaminobenzaldehyde, and for dna content using the picogreen dsdna quantification kit . Ohp content was converted to collagen with a ratio of 1: 7.14 (ohp: collagen). Paraformaldehyde - fixed sections were stained for cell nuclei (40,6-diamidino-2-phenylindole, dapi), glycoasaminoglycans (alcian blue) and collagen (picrosirius red). Dapi - stained sections were visualized at 20 on a nikon t30 inverted fluorescent microscope . Quantitative polarized light microscopy was performed on picrosirius red stained sections to quantify collagen alignment as described previously . Briefly, grayscale images were collected (20) at 10 increments using a green bandpass filter (bp 546 nm) with crossed analyzer and polarizer coordinately rotated through 90. this was repeated with the filter replaced by a compensator . Custom software was then used to determine collagen fiber orientations for a series of nodes within the central portion of each region of interest . Nanofibrous scaffolds of approximately 1 mm thickness were prepared as above and excised along the fiber direction . Two weeks of after seeding with mscs, samples (n=5) were placed in apposition with a 20 mm overlap and secured with porous polypropylene and foil and cultured as above . Lap tests were performed by gripping the overhang on either end of the bilayer and extending to failure at 0.2 mm / s . Aligned nanofibrous scaffolds of approximately 1 mm thickness were excised at 30 from the fiber direction . Agarose was dissolved in pbs at 2, 4, 5, and 6% w / v and melted by autoclaving . Molten agarose was applied between layers of scaffold, and allowed to set at room temperature . No significant difference was observed in cross sectional area across all concentrations, indicating controlled, reproducible interface formation . Significance was established by p 0.05 as determined by two - way anova with a tukey's post hoc test for independent variables of bilayer orientation (parallel / opposing) and culture duration (or agarose concentration). Briefly, poly(-caprolactone) (pcl) were dissolved at 143 mg / ml in equal parts tetrahydrofuran and n, n - dimethylformamide, then extruded at 2.5 ml / h through a spinneret charged to + 13 kv . The resulting nanofibrous jet was collected on a grounded mandrel rotating at 10 m / s and located 20 cm from the spinneret . Aluminum shields on either side of the spinneret were charged to + 9 kv to focus the jet . The spinneret was fanned back and forth to ensure uniform fiber deposition . Mscs were isolated from femoral and tibial bone marrow of 3 - 6 month old calves and expanded to passage 2 as described previously . Scaffolds were hydrated by sequential washes in 100%, 70%, 50% and 30% ethanol and finally phosphate buffered saline (pbs). Before seeding, 50 l of cell solution (1 10 cells / ml) were applied to one side, followed by incubation at 37c for one hour . Scaffolds were then turned and an additional 50 l of cell solution applied to the other side . After two hours further incubation, samples were transferred to chemically defined media (dmem, 0.1m dexamethasone, 40 g / ml lproline, 100 g / ml sodium pyruvate, 1% insulin, transferrin, selenium / premix, and 1% penicillin, streptomycin and fungizone supplemented with 10 ng / ml transforming growth factor 3). Media was replaced twice weekly for the duration of the study . After measuring cross - sectional area using a custom laser device, samples (n=5) were clamped with serrated grips and loaded into an instron 5542 testing device . All testing was performed in a pbs bath . The mechanical testing protocol consisted of: (1) a nominal tare load of 0.1n applied at 0.1% strain / sec, followed by stress relaxation for 5 minutes, (2) 15 preconditioning cycles to 0.1% strain at 0.05% strain / sec, and (3) a quasi - static elongation at 0.1% strain / sec until failure . Strain was determined as extension normalized to gauge length; stress was computed as the load normalized to initial cross - sectional area . Modulus was computed as the slope of the stress - strain plot, determined by regression to the linear portion of the curve . For biochemical analyses, samples were digested for 16 hours in papain at 60c, then analyzed for s - gag content using the 1,9-dimethylmethylene blue dye - binding assay, for orthohydroxyproline (ohp) content (after acid hydrolysis) to determine collagen content by reaction with chloramine t and dimethylaminobenzaldehyde, and for dna content using the picogreen dsdna quantification kit . Ohp content was converted to collagen with a ratio of 1: 7.14 (ohp: collagen). Paraformaldehyde - fixed sections were stained for cell nuclei (40,6-diamidino-2-phenylindole, dapi), glycoasaminoglycans (alcian blue) and collagen (picrosirius red). Dapi - stained sections were visualized at 20 on a nikon t30 inverted fluorescent microscope . Quantitative polarized light microscopy was performed on picrosirius red stained sections to quantify collagen alignment as described previously . Briefly, grayscale images were collected (20) at 10 increments using a green bandpass filter (bp 546 nm) with crossed analyzer and polarizer coordinately rotated through 90. this was repeated with the filter replaced by a compensator . Custom software was then used to determine collagen fiber orientations for a series of nodes within the central portion of each region of interest . Nanofibrous scaffolds of approximately 1 mm thickness were prepared as above and excised along the fiber direction . Two weeks of after seeding with mscs, samples (n=5) were placed in apposition with a 20 mm overlap and secured with porous polypropylene and foil and cultured as above . Lap tests were performed by gripping the overhang on either end of the bilayer and extending to failure at 0.2 mm / s . Aligned nanofibrous scaffolds of approximately 1 mm thickness were excised at 30 from the fiber direction . Agarose was dissolved in pbs at 2, 4, 5, and 6% w / v and melted by autoclaving . Molten agarose was applied between layers of scaffold, and allowed to set at room temperature . No significant difference was observed in cross sectional area across all concentrations, indicating controlled, reproducible interface formation . Significance was established by p 0.05 as determined by two - way anova with a tukey's post hoc test for independent variables of bilayer orientation (parallel / opposing) and culture duration (or agarose concentration).
Cleft lip and palate (clp) is one of the most common craniofacial anomalies of humans . Varied incidence of clp deformity caused due to unknown precise etiology is reported in the literature with severity ranging from minor notching of lip or bifid uvula to complete unilateral or bilateral cleft of the lip and palate . Newborns with abnormal oro - nasal communication face the threat to life due to difficulty in feeding . Hence, there arises an acute demand of feeding appliance that serves as an obturator in the cleft area, in view of the complications of feeding tube . A feeding appliance or obturator is a prosthetic aid that is used to restore the separation between oral and nasal cavities . In addition, it is also the primary step in the fabrication of different devices such as palatal obturator, articulation development prosthesis, palatopharyngeal obturator, palatal lift prosthesis, nasal conformer, and appliances for presurgical infant orthopedic or presurgical nasoalveolar molding, those that are introduced to enhance speech, esthetics, and function . It has been emphasized in literature that every precaution must be taken while making impression for neonates and infants with cleft palate, and surgeons' presence is needed to manage airway emergency . Maneuvering the child's head position forward to avoid the aspiration of impression material has been the only suggested precaution . This does not ensure complete control over the impression material and avoidance of complication of impression material being pushed too deep into the nasal cavity or distally through the pharynx . This clinical report presents dental putty - gauge hybrid impression technique as a simple and safe method of maxillary impression making in neonates and infants with cleft palate, permitting complete control over impression material during functional movements . On extraoral examination, there was bilateral complete cleft lip with forward placed premaxilla [figure 1]. Intraoral examination revealed a median cleft of the palate involving complete soft and hard palate [figure 2]. They were explained the treatment plan for fabrication of feeding plate and consent was obtained . Extraoral view showing bilateral cleft lip intraoral view showing cleft lip and palate it was planned to make the maxillary impression when the infant was fully awake, without any premedication or anesthesia . The infant was seated upright in his mother's lap in dental chair with face forward . Small size of the mouth opening did not permit the use of impression tray or flat wooden stick as carrier for the material . Thus, it was planned to make impression with finger - loaded impression material . Fast setting elastomeric putty impression material (express std, 3 m espe, usa) was mixed and loaded on the index finger and an adequately large piece of gauze was wrapped all over the loaded impression material with loose ends wrapped toward the hand [figures 3 and 4]. The gauze permitted control over the material and restricted the excessive displacement of impression material toward the nasal cavity and pharynx [figure 5]. Thick consistency of the impression material did not permit leakage of material through the gauze and kept it within the flexible confines of the gauze . The resultant sucking pressure embedded the gauze threads in the putty impression material as the easily moldable gauge did not offer any resistance . After the impression material was set, the impression was removed in toto from the patient's mouth [figures 6 and 7]. Then, the impression was poured in dental stone (kalstone, kalabhai karson pvt ltd)., mumbai, maharashtra, india) and the master cast was obtained [figure 8]. Single visit feeding obturator was fabricated using clear, flexible thermal - forming material with a midline extraoral extension for a hole to tie a safety thread [figure 9]. Dental putty loaded on the index finger gauze wrapped around the putty impression material loaded on the finger maxillary impression made with functional movements by neonate hybrid impression retrieved in toto dental putty maxillary impression clear soft feeding obturator with safety thread attached feeding obturator in situ parents were demonstrated about the use and hygiene maintenance of the feeding plate . A regular follow - up after 24 h and monthly follow - ups later were scheduled and the parents were advised to use the appliance till surgical intervention . A cleft palate hampers intraoral pressure built - up required for suction and favors nasal reflex of food, thus posing challenge of middle ear infections . Feeding is an immediate concern for newborn with clp where the role of dentist and the feeding plate is evident . Various head and body positions and diverse impression materials have been used to make maxillary impression in neonates and infants with suggestions to take all precautions including the presence of surgeon to handle airway emergency . A feeding appliance bridges the gap between oral and nasal cavities . Feeding obturator becomes urgent in clp infants considering the health of the infant as surgical treatment usually starts at 23 months of age . However, little is found in literature regarding the impression required to fabricate these maxillary appliances . Impression making is the initial, crucial, and clinical step in cleft palate of infants and poses a challenge because of lack of cooperation from patient, inadequate size of oral cavity, and nonavailability of suitable impression trays . There is a risk of breakage of some parts of impression material that may get lodged in the undercut in the defect or the child can swallow or aspirate the material . Difficulty in withdrawal of impression, cyanotic events, and asphyxiation has been encountered during impression making . Displaced intranasal impression material may remain undetected for a very long period of time . Impression compound softened and placed on finger is the commonly used method to make preliminary impression . Individualized impression trays for clp have been designed and the tray handle has also been used to carry the material . Impression making under general anesthesia has been adopted for infants . Adequate suctioning and resuscitation facilities should be available chairside . In case of airway emergency, it is advised to maneuver the infant position and head position to keep the tongue forward to avoid posterior displacement of impression material . Making the child to cry throughout the impression procedure however, it does not prevent the thin consistency material passing down the pharynx . In this case, the child was not crying during the procedure, but performed the functional movements to mold the impression material to obtain accurate anatomic details . Cleft palate affects feeding, facial growth, dentition development, speech, and may cause psychological problems for the child and parents as acceptance of the parents for the dysmorphic child can be difficult . However, in the present case, parents were involved in the care from the start of therapy that provided assurance and motivation to them . A variety of impression materials have been advocated; however, putty type vinyl polysiloxane has been used in this case because its high viscosity reduces the risk of aspiration and there is no risk of thermal injury to the delicate oral mucosa of the neonate unlike that with the use of wax or thermoplastic impression compound . The use of gauze wrapped around the impression material throughout the functional movements completely removed the chances of aspiration or swallowing of material making, it a risk - free procedure in an awake child as young as 1-day - old . However, in this patient, soft, clear, and flexible thermoplastic material was used as it permitted a single - step appliance fabrication . The feeding plate was delivered without any delay, on the same day in view of the urgency for feeding the newborn . The prosthetic therapy delivered through feeding plate remains a suitable treatment modality until the time prenatal diagnosis and intrauterine fetal surgery through feto - endoscopic approach for human fetus with a clp are well established . This article describes the successful immediate oral rehabilitation of clp neonate achieved with simple feeding appliance and risk - free procedure that decreased the stress for the parents and the child and provided instant feeding ability . Maxillary impression procedure in infants with cleft palate poses a unique set of challenges and we should be well - equipped for every precaution while making impression . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Mammals are the only animals that secrete a complex fluid from an elaborated skin gland to provide both innate protection and nourishment for their newborn . There are more than 4,000 species of mammals with striking similarities in the structure and function of their mammary glands as well as in their unique milk components such as the caseins, -lactalbumin, lactoferrin, lactose, and milk fat . Nevertheless, variations are exhibited in the arrangement and numbers of mammary gland, milk composition, and suckling strategies . Mammary gland development begins at puberty and is maintained throughout pregnancy until lactation . During these last stages, development compromises numerous overlapping programs such as branching morphogenesis, inductive stromal - epithelial interactions, programmed cell death, extracellular matrix remodeling, and hormone action . Current knowledge of the molecular regulation of mammary development and lactation has largely been derived from the dissection of signaling networks in cell culture systems and phenotypic characterization of genetically altered mice as well as genomewide approaches such as microarrays . Nonetheless, to date, regulation of mammary gland development during pregnancy and lactation is incompletely understood . Lactation is regarded as one of the most remarkable products of evolution whose processes include the development of mammary tissue as well as the synthesis and secretion of milk . Consequently, despite the fact that the development of mammary tissue and the synthesis and secretion of milk are considered as complex dynamic physiological processes, both must preserve overall common characteristics among mammals . Considering the underlying assumption that important biological functions are often conserved across species, genes expressed across multiple species are likely to have conserved functions . Given the completion of the dna sequence of the human, mouse, and rat genomes, genes identified in microarray studies can be readily compared across species with respect to orthologous genes . Therefore, a cross - species hybridization (csh) experiment could provide significant information concerning probable conserved gene networks among mammals . In a csh experiment, there is hybridization of rna from one or more (target) species to a microarray that contains dna (cdna or oligomers) from another (reference) species and represents a valuable tool for the identification of orthologous genes . Thus, a csh microarray analysis offers the possibility of furthering our understanding of cross - species commonalities and differences that could lead to more effective use of animal models to understand the regulation of mammary gland development at the molecular level . Dissection of unique patterns of expression of orthologous clusters of genes among species throughout distinct physiological time points along pregnancy and lactation could prove useful in the integrative analysis of the information available for discerning the molecular events underlying the regulation of mammary gland development and function that lead to milk synthesis . In this study, these data resulted from heterologous microarrays of target rna samples derived from rat mammary gland during distinct stages of pregnancy and lactation in order to extrapolate and enhance the understanding on transcriptional module networks or coregulated functional gene groups conserved in rodents and in the development of the mammary gland in humans . Fifteen female sprague dawley rats were obtained from the animal care facility of centro mdico nacional siglo xxi of the mexican institute of social security (imss) in mexico city . Animals were housed at 22 2c with a 12 h light / dark cycle with free access to water, and a purified diet was administered ad libitum during pregnancy and lactation ., rats were randomly assigned to five groups representing distinct time points in mammary gland development: virgin (v), day 5 (p5) and, day 14 (p14) of pregnancy and day 1 (l1), and day 12 (l12) of lactation . The day on which sperm was identified in vaginal smears was designated as day 1 of pregnancy and the day of parturition was designated as day 1 of lactation . Rats were euthanized, and whole mammary tissue was removed from v, p5, p14, l1, and l12 rats . Tissue was immediately frozen in liquid nitrogen and stored at 70c for subsequent total rna isolation or histological analysis . Total rna was isolated from tissue (0.1 - 0.2 g) using trizol (invitrogen, carlsbad, ca, usa) following the method of chomczynski and sacchi . Total rna from mammary tissue was isolated from three different animals of each physiological period (v, p5, p14, l1, and l12), pooled, and kept in aliquots for later determination of purity and integrity . Four microarray datasets generated using the custom - designed mus musculus oligonucleotide array containing 65-mer probe sets representing 22,000 transcripts (microarray unit, cellular physiology institute, unam, mexico city) were analyzed . Each dataset represented distinct time points in mammary gland development such as p5, p14, l1, and l12 . Histologically, the mammary proliferative stage is represented by p5, the secretory differentiation stage by p14, early lactation by l1, and full lactation by l12 . Design of the microarray experiments is presented in table s1 in supplementary materials available online at http://dx.doi.org/10.1155/2013/624681 . Ten g of total pooled rna was reverse transcribed into cdna incorporating dutp - cy3 or dutp - cy5 and using the cyscribe first - strand cdna labeling kit (amersham biosciences, piscataway, nj, usa). Sunnyvale, ca, usa), equal quantities of labeled cdna were hybridized to the m22 k_01 microarray for 14 h at 42c . Four hybridization assays were carried out as follows: (a) the fluorophore used was dutp - cy3 for control nonpregnant virgin rats (v) and dutp - cy5 for p5, (b) dutp - cy3 for v and dutp - cy5 for p14, (c) dutp - cy3 for v and dutp - cy5 for l1, and (d) dutp - cy3 for v and dutp - cy5 for l12 . Data acquisition and analysis of array images were performed in scanarray 4000 with its accompanying software scanarray 4000 from packard biochips . Microarray data analysis was performed with free software genarise, which was developed in the computing unit of the cellular physiology institute of the unam (http://www.ifc.unam.mx/genarise/). The goal of genarise is to identify which genes show good evidence of being differentially expressed . The software identifies differentially expressed genes by calculating an intensity - dependent z - score . Elements with a z - score> 1.5 standard deviations are considered to be significantly and differentially expressed genes . The complete set of raw excel data files have been deposited at gene expression omnibus (geo) and are available on the geo website (i d geo gse22545). Gene lists were generated by a set of multiple comparisons among the distinct developmental stages and intersection in venn diagrams . Two - way hierarchical clustering with average linkage and a range of 5 to 15 k - means classifications were used to group our time series data using open source software cluster v3.0 . Java treeview was used to display the clustering results as dendogram or heat map representations . We adopted the procedure as described in to code the mean expression of a cluster at each stage as flat, decreased, and increased and converted it to numerical representation . Putative orthologous genes in rat, mouse, and human were identified from a genome comparative search with roundup (http://rodeo.med.harvard.edu/tools/roundup). Roundup is an ortholog and phylogenetic profile retrieval tool backed by a massive repository of orthologous and associated evolutionary distances that were built using the reciprocal smallest distance algorithm . The search was done with a stringent blast e - value threshold of 1.0 10 and a divergence threshold of 0.2 . The david 2.0 functional annotation tool (http://david.abcc.ncifcrf.gov/summary.jsp) was used to sort and arrange the similar, redundant, and heterogeneous annotation contents from a set of genes into defined functional groups . In the case of insufficient gene ontology information, published pathway mapping was accomplished using the kyoto encyclopedia of genes and genomes (kegg) database of biological systems that integrates genomic, chemical, and systemic functional information (http://www.genome.jp/kegg/kegg2.html). Gene lists were converted to human swissprot ids using tables from the ensembl database, release 42 . For each list of human swissprot ids, interactions between those gene products were obtained from online predicted human interaction database (ophid) and postprocessed using custom scripts to determine all linkages in the network and to generate a network file . This network file was then explored using navigator v2.0.15, a program for large network analysis (http://ophid.utoronto.ca/navigator/index.html). Transcription - factor - binding site (tfbs) prediction was accomplished using core_tf (conserved and over - represented transcription factor binding sites), a web - based tool that identifies overrepresented tfbs in promoters from coexpressed genes aided by the evaluation of cross - species conservation . We measured the relative transcript levels of 14 target genes, and five genes commonly used as references such as glyceraldehyde-3-phosphate dehydrogenase (gapdh), beta - actin (actb), and ribosomal large protein p0 (rlp0) were used as high abundance internal controls as well as splicing factor arginine / serine - rich 1 (sfrs1) and hypoxanthine guanine phosphoribosyl transferase 1 (hprt1) as medium- and low - abundance internal controls, respectively (table s2). Quantitative real - time pcr was performed in a 20 l reaction with 5.0 l from 1/4 reverse transcription dilution using the lightcycler probes master mix (roche diagnostics, mannheim, germany) containing 0.2 m of mrna - specific primers and 0.1 m corresponding upl probe into lightcycler microplate wells under reduced light conditions . The results are expressed as mean sem of at least three individual experimental observations . Statistically significant differences among experimental groups (between the mean values of each group) were determined by an unpaired students t - test (z - test), anova, or a modified fisher's exact test . The rat mammary gland undergoes a series of dramatic phenotypic changes during pregnancy and lactation . In order to determine the integrity of the dissected inguinal mammary glands, a gross histological evaluation of the characteristic cytomorphological features four time points (pregnancy days 5 and 14; lactation days 1 and 12) were selected to represent distinct periods in mammary gland development . Histologically, the mammary proliferative stage is represented by p5, the secretory differentiation stage by p14, early lactation by l1, and full lactation by l12 . As reported elsewhere, initial changes observed during pregnancy include an increase in ductal branching and the formation of alveolar buds (figures 1(c) and 1(d)). The latter half of pregnancy is characterized by the expansion of alveolar buds to form clusters of lobuloalveolar units followed by the differentiation of these structures into presecretory structures . By day 14 of pregnancy, there is a readily apparent increase in the size of the epithelial compartment (ep) (figures 1(e) and 1(f)), and expansion of the epithelium (whereas the adipose compartment decreases) continues until the epithelial compartment predominates by onset of lactation (figures 1(g) and 1(h)). By day 12 of as expected, examination of the histology of the mammary gland at this stage reveals prominent luminal structures (l) and ducts and few adipocytes visible at this time (figures 1(i) and 1(j)). In this study, we analyzed expression profiles of 22,000 transcripts using murine microarrays and rna samples of mg from virgin, pregnant, and lactating rats by cross - species hybridization . We first identified the total number of genes differentially expressed throughout distinct time points in mammary gland development such as p5, p14, l1, and l12 . A total of 807 oligonucleotide probe sets representing 521 annotated genes showed differential expression in at least one of four physiological time points evaluated, taking into consideration a mean z - score cutoff value of 1.50 standard deviations using genarise . During early pregnancy (day 5), most of these transcripts (123, 77.8%) were upregulated, suggesting a feasible tendency in the direction of gain of function versus the virgin stage (v). Likewise, in mid - pregnancy (day 14), as opposed to the virgin stage, the number of transcripts with an altered expression maintained a similar value (133 transcripts; 89.26% upregulated). During early and mid - lactation (days 1 and 12), 342 and 461 transcripts were differentially expressed, corresponding to a percentage of 64.0 and 38.4 overexpressed, respectively (figure 2(a)). To further illustrate the differences and commonalities among the four physiological time points, changes in gene expression were also interpreted with a venn diagram . As shown in figure 2(b), the descriptive table of the venn diagram denotes the number of genes showing upregulation () or downregulation () uniquely at pregnancy (day 5 or 14) or lactation (day 1 or 12) and differential expression at a combination of stages . Venn diagram analysis indicated that 47.2% (381/807) of all the differentially expressed transcripts presented an average significant z - score fold change (z> 1.5) exclusively during either or both time points of lactation . Interestingly, among the 381 altered gene transcripts during lactation, 64.8% (247/381) were found downregulated, implying as previously stated by lemay et al . (2007) that mammary epithelial cells become biofactories not by gain of function but by a broad suppression of function to effectively push all cell resources towards a very few important tasks . All the gene sets that shared spatial and temporal distributions (overlapping changes in expression) are listed in additional data files (table s3). To determine global alterations in gene expression across developmental stages of the mammary gland from early pregnancy to mid - lactation, we performed a complete - linkage hierarchical clustering with an euclidean distance similarity metric on the expression profiles of the differentially expressed genes (annotated and est) across all four time points . The expression profiles of the 807 genetic elements resulted in six predominant clusters on a dendogram (designated clusters 16 in figure 3). This major trend is a decline in gene expression during mid - pregnancy that remains low during lactation . Cluster 2 (c2) describes 25.77% of the total data population and is characterized by a linear decrement in gene expression towards mid - lactation . However, positive z - score values are retained with respect to the reference stage (virgin). The remainder of the clusters (c3c6) appears to explain between 3.09 and 17.84% of the data variation . In c3 and c4, gene expression rises exponentially from early pregnancy, reaching a plateau during mid - lactation . However, the slope of the curve is even steeper in c3 in comparison to c4 . In cluster 5 (c5), 65 elements matched the profile outline (inverted sigmoid form) of major trend c1 although the reduction tendency was less marked . In cluster 6 (c6), expression was roughly unchanged during pregnancy and lactation . Even so, the relative abundance of transcripts remained in a higher proportion than the reference virgin stage as described for c2 . This transcriptional profile, involved in the mammary development program identified in rat, could be conserved in others mammals like mouse . Consequently, in order to delineate potential groups of coregulated genes, final cluster membership was determined by a k - means analysis based on the preestimated number (six) of gene clusters . K - means clustering revealed six distinct clusters (k16) that distinguished up from down, early from middle, and transient from sustained changes in expression (figure 4; table s5). Each of the six clusters was designated with its unique trajectory expression profile signature across stages (pregnancy days 5 and 14, lactation days 1 and 12) as presented in figure 4 . There were two major groups of 245 under- and 175 overexpressed tags during lactation only (table s5, k1: 1,1,0,0 and k3: 1,1,2,2 according to the procedure of rudolph et al ., to code the mean expression, see section 2). Among the typical upregulated genes of lactation stage are the milk protein (casein alpha (csn1s1, csn1s2a), casein beta (csn2), and whey acidic protein (wap)) and biogenesis genes that mainly concern glucose and lipid metabolism (akr1c6, aldob, ugt2b1, plb1, apoe, and sult2b1) and transcriptional regulation (stat5a, pou2af1). Among those genes found significantly downregulated only during lactation, several play an important role in the regulation of apoptosis, mediation of metastatic behavior (epithelial - mesenchymal transition), or ubiquitin - mediated protein catabolism (lysosome degradation) in the mammary gland including igfbp5, mmp2, and ube2r2 [14, 15]. One hundred forty - nine genes were upregulated exclusively during early pregnancy (k2: 2,1,1,1) such as esr1, esr2, tshr, and oxt . These participate in the transduction of hormonal status [2, 16] involved in the modulation of important physiological processes such as carbohydrate metabolism (creb3l4, hk1, and coasy); glutathione metabolism (ggt1, mgst1, and gstm6); cell differentiation (foxa1, mtap7, gdf1, twist2, hey1, dll4, and pcaf) [1923], stromal - epithelial communication (cell - cell junctions) (cldn10, mpp5, and epb4), and cell adhesion (matn1, krt71, mpzl2, and dscaml1). The smallest group of 24 genes were significantly upregulated exclusively at the onset of lactation (k4: 1,1,2,1) such as lpo, cd8a, and irs1, important for lactogenesis, particularly in milk production capabilities and related immunotropic constituents (antigen - specific cd8 + t cells) found in colostrum [26, 27]. For example, acta1, flnc, and pax7, which are either restricted to muscular tissues or involved in myogenic development and cellular differentiation [28, 29], are included in this group . According to the trajectory profile signature, 95 additional genes were found upregulated at all stages evaluated (k6: 2,2,2,2). Interestingly, most of the overexpressed genes in this group include general transcription and translation (including spliceosome and protein folding) machinery factors (eif4a2, eif2ak1, etf1, taf1, ercc2, sart1, ppih, and dbr1) [30, 31] as well as structural (itga5, actg1, add2, cldnd2, rptn, and triobp) and basal metabolic genes (pank4, agpat5, cyp24a1, and phyh). Once the gene clusters were properly defined, identification of orthologous gene transcripts among the time course differentially expressed gene list subsets was critical for reliable comparison of gene function and subsequent determination of probably conserved transcriptional modules implicated in biological processes during the development of mammary tissue . According to genome comparative roundup orthologous database of a total of 448 transcripts upregulated and 371 downregulated, 213 (upregulated) and 183 (downregulated) genes were identified as orthologous to rat and/or human . The remainder of the genes was discarded or removed from subsequent analysis due to lack of similarity, insufficient information, or unknown identifiers . A complete list of orthologous genes from each dataset was compiled (table s6). Among the upregulated orthologous genes to rat and/or human are those encoding to milk proteins, carbohydrate and lipid metabolism, transcriptional factors [17, 34], transduction of hormones, glutathione metabolism, and cell differentiation [19, 20]. Others are associated with stromal - epithelial communication, cell adhesion, lactogenesis, and general transcription and translation machinery factors as well as structural and basal metabolic genes [3335]. Among those genes found significantly downregulated, several play an important role in the regulation of apoptosis, mediation of metastatic behavior (epithelial - mesenchymal transition), or ubiquitin - mediated protein catabolism (lysosome degradation) [14, 15]. Also, genes restricted to muscular tissues or involved in myogenic development and cellular differentiation [28, 36] are downregulated . Taking into consideration their temporal expression profile signature and the fact that they represent different k - means cluster, 14 genes were selected for real - time pcr analysis (table s2). Results show that the expression trends were consistent with the results from the microarray analysis . Correlation analysis showed good agreement between real - time rt - pcr and microarray analysis . Microarray results for all 14 genes tested were confirmed by real - time rt - pcr with regard to direction and significance of change (figure 5). Conservation of functional blocks of genes is likely to be more important in a cross - species comparison . We found distinct blocks of significantly differentially expressed genes within different cytogenetic regions of the rat with homologous chromosomal segments in human and mouse . However, human, mouse, and rat have different chromosomal arrangements . Genes in these blocks appear in contiguous cytogenetic regions, irrespective of species and chromosomal location . This finding is not surprising considering the close evolutionary distance between the species where 278 orthologous segments are reported to be shared between human and rat, and 280 segments are reported to be shared between human and mouse . It is proposed that these gene blocks may be significant for mammary gland development and maintenance and progression of lactation across human, rat, and mouse . For example, genes in the blocks may be coordinately expressed to share transcription programs as stated in previous studies . The argument may be made against the feasibility of using rodent data to draw inferences to human mammary gland gene expression . However, our objective in this study was to utilize the best available sources of information such as rat gene expression data during mammary development and mapping data to develop hypotheses on putative functional gene blocks conserved across species, underlying similarities despite reported differences in the architecture and hormonal control of mammary glands between rats and other rodents and between rats and humans [38, 39]. In an effort to further characterize potential highly coregulated gene blocks, we combined transcription - factor binding - site prediction along the promoters of each gene member with the detection of expression profiles of annotated altered transcripts categorized as nucleic acid binding protein . Several families of transcription factors were identified (table s7). For the most part, the presence of cis - elements found with core_tf (http://www.lgtc.nl/core_tf) in the promoters of the genes slc44a4, ppt2, and b3galt4 that compromises the conserved block d15 (table s8) along with the cotranscription of mrnas that encoded for trans - regulator elements suggests that they are most likely modulated by transcription factors runx2, creb3l4, pou3f2, and pou2af1 . Correspondingly, the gene members of block u1 may possibly be coregulated by transcription factors stat5a, foxa1, creb3l4, and pou2af1 . In the same manner, other gene blocks (u3, u8, u9, d1, d9, and d14, table s8) were found most likely co - regulated by a minor number of transcription factors (foxa1, creb3l4, pou2af1, or egr2). Hence, identification of conserved overrepresented upstream motifs unravels putative regulatory elements for transcription (figure 6) in at least half of the gene block members reported in this study . Consequently, these results strongly substantiate the maintenance of comparable transcriptional regulation programs among the predicted coexpressed modules . Because cotranscription of genes in conserved blocks may allow concerted expression of gene products involved in the same response or pathway, integration of this type of analysis thus, the co - regulated clusters we proposed may indeed be conserved transcriptional modules through evolution, at least between rodents and primates . Heterologous hybridization experiments on any microarray are of limited use for genes that have undergone rapid evolutionary change in coding regions, large rearrangements, and duplication . Long oligonucleotide - based microarray platform may be more suitable for cross - species gene expression studies than a short oligonucleotide - based system . This comparative approach is based on the assumption that similar gene sequences in closely related species allow a reasonably reliable detection of many orthologous genes . For instance, according to several independent and unrelated studies carried out on comparable 50 to 60-mer oligonucleotide microarrays, cross - hybridization was observed only with genes with 50%75% overall sequence identity, respectively [44, 45]. Considering that orthologous genes between human and mouse and between human and rat both have a mean of ~85% sequence identity, validity of the results obtained in this study despite the problems encountered by csh in comparison to ssh seems upholding . In fact, the nucleotide sequence alignment confirmed an> 75.3% homology at least for the transcript members of the distinct gene blocks described, depending on the sequence evaluated among primates and rodents reinforcing the notion of attaining valid biological results . In addition, similar expression trends for distinct probe sets for one corresponding gene (data not shown) seem to largely substantiate the certainty and reproducibility of hybridization results obtained in this study . Because of the challenges inherent to csh data, their confirmation by other techniques is essential . In addition to qrt - pcr, orthologous gene expression profiles with syntenic regions of rat, mouse, and human chromosomes reinforce another confirmation method that potentially substantiates the csh results obtained in this study . Nonetheless, further validation of the results must be carried out by using csh of human rna to mouse oligonucleotide arrays . This study provides access to a prevalidated platform for analyzing transcriptional changes in rat mammary gland . This paper will hopefully spur an increase of mammary gland csh transcriptome analysis, thus adding to our knowledge base of this interesting evolutionary feature among mammals . However, although we acknowledge the multitude of aspects that can be elucidated by traditional ssh transcriptome analysis, we believe the biggest potential of the presented microarray lies in the multispecies - type studies described . We demonstrated that data analysis strategies such as the combination of orthologous gene expression profiles and chromosome mapping in conjunction with directed promoter transcription - factor binding - site prediction presented here can add strength to conclusions and help identify systems and responses that are conserved across the mammal taxa . The possibility of studying the evolutionary depth of transcriptional regulation adds a new dimension to comparative transcriptomic, particularly identification of differentially co - regulated gene blocks mapped to highly conserved syntenic chromosomal regions, which is important in mammary gland development using csh experiments among mammal species.
When a body part is under pressure, tissue cells do not receive nutrients properly, thereby resulting in their death and the appearance of a lesion . In addition to exposure to pressure, an elevated body temperature and a high skin humidity may also contribute to pressure ulcer formation, which appears as an irritation in the upper layers of the skin and may reach muscles and bone tissue2 . Pressure ulcers are harmful to subjects, particularly those who have additional risk factors related to poor health3 . In this sense, therefore, it is important to prevent pressure ulcers . The correlation between being seated and the presence of pressure ulcers has been reported frequently since epidemiological studies of pressure ulcers were first conducted4 . Barbenelet et al . Reported the pressure ulcer prevalence survey in the united kingdom and noted that chair - bound patients consistently exhibited a higher frequency of developing pressure sores compared with bed - bound patients with a similar degree of helplessness5 . Other surveys have also shown this association between being seated and the presence of pressure ulcers6, 7 . The guidelines concerning pressure ulcers issued by the national institute for health and care excellence (nice) in the united kingdom include a statement regarding seating, in which the authors stressed the need for qualified assessment of seating needs, the importance of correct eating positions, and the need to maintain posture and support the feet when using a wheelchair8 . In people who are at risk for pressure ulcers or in those with bedsores, pressure redistribution must be tended to such that no pressure is applied to the pressure ulcer9 . A number of management strategies, including a number of commercially available alternating pressure cushions and wedges, can provide pressure redistribution10, 11 . Lee reported the effectiveness of cushions and an anterior wedge sitting position in pressure ulcers . In their study, they showed that the type of cushion and appropriate height wedges made a statistically significant difference in sitting pressure12 . Several studies found a correlation between cushion types and sitting pressure or between anterior wedge height and sitting pressure . However, pressure redistribution when cushions and wedges are applied simultaneously has not been investigated . Consequently, the purpose of this study was to analyze user cushion interface pressure redistribution when different cushion types and height anterior wedges are applied simultaneously for healthy volunteers . This study was a pilot study to direct future research, to perform adult norm pressure mapping . All participants were identified as able - bodied subjects, and the procedure was fully explained to them . The exclusion criteria included any type of sitting problems, or hearing, visual, or cognitive impairments that interfere with accurate participant assessments . The subjects were informed that they could withdraw from the study at any stage for whatever reason . Participant characteristics are provided in table 1table 1.general characteristics of the subjectsmale (n=15)female (n=21)age (years)21.7 2.020.1 0.5weight (kg)66.1 8.753.9 9.2height (cm)171.8 6.2158.4 5.1seat to footplate (cm)46.7 5.343.8 4.6seat depth (cm)49.5 3.545.9 4.9seat width (cm)36.0 3.434.3 3.6hip ()99.9 5.599.5 7.2knee ()93.9 5.999.5 6.8ankle ()93.4 5.797.3 6.3 . The conformat system and research software v.7.2 were used for pressure mapping and data acquisition, respectively . One occupational therapist and three university students collected the pressure data while the participants were sitting on a firm surface without a cushion, a 5-cm height gel cushion, a 5-cm height memory foam cushion, a 3-cm anterior wedge without cushion, a 3-cm anterior wedge with a 5-cm height gel cushion, a 3-cm anterior wedge with a 5-cm height memory foam cushion, a 6-cm anterior wedge without cushion, a 6-cm anterior wedge with a 5-cm height gel cushion, and a 6-cm anterior wedge with a 5-cm height memory foam cushion . The participants were instructed to keep their chins tucked in, spines straight, hands on their thighs, and pelvis neutrally positioned when sitting on the cushions . They were initially instructed to flex their hips, knees, and ankles at approximately 90 and to put their feet flat on the floor . Each joint angle and sitting position were checked for each measurement . After measurement, the pressure map was divided into four quadrants on screen graphics, with each quadrant representing the left hip, left thigh, right hip, and right thigh . For the quadrant division, the mergl method was adapted and used to analyze the sitting pressure on the cushion . The peak pressure was the mean of the maximum pressures measured with four sensors in each quadrant . The mean pressure was the mean of the pressures measured with every sensors in each quadrant . The participant characteristics were tested by descriptive statistical analysis, and results were tested by a one - way analysis of variance and post - hoc analysis . General demographic information, including age, weight, height, and sitting position, is shown in table 1 . In both genders, the mean and peak pressures were the lowest when sitting on a 5-cm foam cushion . The mean pressure in the hip and thigh was the highest on a 6-cm wedge and on a firm surface, respectively . The mean pressure was observed to increase in the following order: a foam cushion, a 3-cm anterior wedge with a 5-cm foam cushion, a 6-cm anterior wedge with a 5-cm foam cushion, a 5-cm gel cushion, a 3-cm anterior wedge with a 5-cm gel cushion, a 6-cm anterior wedge with a 5-cm gel cushion, a 3-cm anterior wedge without cushion, without cushion, and a 6-cm anterior wedge without cushion (tables 2table 2.peak pressure distribution according to wedge height and cushion type (n=36, mmhg)firm surface5 cm gel cushion5 cm foam cushion3 cm wedge5 cm gel cushion with 3 cm wedge5 cm foam cushion with 3 cm wedge6 cm wedge5 cm gel cushion with 6 cm wedge5 cm foam cushion with 6 cm wedgerhpp304.43 95.46123.04 71.21116.09 69.57308.64 91.46131.88 55.87122.25 67.42346.25 91.69158.92 55.58168.22 43.09rtpp62.83 19.9352.49 17.7943.85 24.2877.73 29.5757.01 21.3948.19 18.1585.52 39.8966.28 25.9465.20 27.81lhpp305.89 85.11120.03 40.14127.96 42.27321.21 88.04130.64 57.14130.03 92.82321.91 94.82146.31 65.99166.83 88.00ltpp71.47 25.2454.61 24.8141.93 17.0567.99 37.9858.01 24.6143.75 17.4792.15 38.3757.52 25.0761.00 21.22r / lhpp: right / left hip pressure peak pressure; r / ltpp: right / left thigh pressure peak, * p<0.05 and 3table 3.mean pressure distribution according to wedge height and cushion type (n=36, mmhg)firm surface5 cm gel cushion5 cm foam cushion3 cm wedge5 cm gel cushion with 3 cm wedge5 cm foam cushion with 3 cm wedge6 cm wedge5 cm gel cushion with 6 cm wedge5 cm foam cushion with 6 cm wedgetpm59.09 10.2937.86 9.2429.45 9.3057.00 11.4939.14 6.5029.60 6.4360.45 10.0441.70 7.1330.92 8.52rhpm85.04 17.6249.76 15.3939.41 13.0484.55 19.6454.74 14.0541.80 12.7288.61 18.3067.02 48.0342.65 15.67rtpm31.88 11.7325.45 12.4117.78 7.1127.04 9.8524.93 6.6818.81 5.5629.26 9.3225.31 7.5822.59 19.51lhpm85.86 16.4349.53 16.4142.53 18.7287.96 17.4967.39 6.0842.58 16.9288.68 17.0258.01 17.4545.83 17.91ltpm30.84 11.1325.92 8.1218.19 6.9227.07 9.3426.61 6.7219.15 5.8129.06 9.6526.43 7.9027.71 8.02tpm: total pressure mean; r / lhpm: right / left hip pressure mean; r / lhpp: right / left hip pressure peak, p<0.05). R / lhpp: right / left hip pressure peak pressure; r / ltpp: right / left thigh pressure peak, * p<0.05 tpm: total pressure mean; r / lhpm: right / left hip pressure mean; r / lhpp: right / left hip pressure peak, * p<0.05 prolonged mechanical loading can lead to breakdown of the skin and underlying tissues, which can, in turn, develop into a pressure ulcer13 . It may be caused by inadequate blood supply and resulting reperfusion injury when blood re - enters the tissue . When sitting in the same position for extended periods, the dull ache experienced is indicative of impeded blood flow to affected areas . The shortage of blood supply may lead to tissue damage and cell death within 2 h14 . One of the assessment factors relating to the prevention of pressure sores is the pressure of cushions on clients15 . The benefits of pressure relief and/or redistribution in minimizing pressure - related health risk (including of pressure ulcer), among other issues, have been well documented . However, few studies have offered information on the combination effects of cushions with anterior wedges . Hence, the objective of this study was to evaluate pressure redistribution effects on the user cushion interface pressure of healthy volunteers in their 20s when applying different cushions, including a firm surface with and without different height anterior wedges . Our results show that the mean and peak pressures were greatest when sitting without a cushion with 3-cm and 6-cm anterior wedges and without anterior wedge compared with the other sitting arrangements . The mean pressure in the hip was the highest when sitting on a 6-cm anterior wedge without a cushion, whereas the mean pressure in the thigh was the highest when sitting on a firm surface without an anterior wedge . In addition, the mean and peak pressures were the lowest when sitting on a foam cushion without an anterior wedge . Gong and an reported that the mean and peak pressures were the lowest when a 6-cm anterior wedge was used . However, in this study, these two variables were the lowest when sitting on a foam cushion without an anterior wedge11 . The participants of this study sit with the hip, knee, and ankle joint at an angle of more than 90. therefore, the sitting professional must assess posture before recommending the use of cushions and wedges . From the results, we presumed that the foam cushion could be the best choice for pressure redistribution . These results suggest that people who sit, particularly those with diseases, seek out professional support in choosing the appropriate cushion and height of their chair based on their needs . The present study only compared the performance of two cushions, including a firm surface, and two types of anterior wedges, in redistributing user hence, the number of participants, cushions, and wedge types, as well as the height of the cushion and wedges, must be expanded for generalization in future studies . Cushion interface pressure redistribution, the relationship between sitting positions and interface pressure redistribution must be investigated . As the proper cushion types and anterior height wedges for people with or without diseases, such as stroke, spinal cord injury, and muscular dystrophy, must be established, this study establishes important guidelines for future research for establishing a sitting environment.
Tropomyosins (tms) are a family of highly conserved actin binding proteins which are expressed in all eukaryotes from yeast to humans . Tms play a critical role in the control of ca - regulated thin filament function in striated muscle contraction . Except for zebrafish, in vertebrates, there are four known tropomyosin (tpm) genes (designated as tpm1, tpm2, tpm3, and tpm4) [16]. In zebrafish, six tropomyosin genes have been reported . Each of the tpm genes generates a multitude of tissue and developmental specific isoforms via alternate splicing . Tpm1 is known to produce at least ten alternatively spliced transcript variants . In mammals, the predominant striated muscle isoform is tpm1 containing exons 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a / b, which encode 284 amino acid residues . Our laboratory first reported another striated muscle isoform known as tpm1 in axolotl heart that contains exons 1a, 2a (instead of exon 2b), 3, 4, 5, 6b, 7, 8, and 9a / b . We also reported the expression of tpm1 in axolotl skeletal muscle, human heart, and embryonic chicken heart . In humans, tpm1 is expressed in both fetal and adult hearts, whereas in chicken both tpm1 and tpm1 are expressed only in embryonic heart . Interestingly, transgenic mice that overexpressed human tpm1, in a cardiac - specific manner, were found to develop a dilated cardiomyopathy - like syndrome . Two possible reasons for observing some abnormalities in tg mouse hearts ectopically overexpressing human tpm1 were that too much tpm1 is toxic to the mouse cardiomyocyte or that human tpm1 is variant from its mouse homologue and, for that reason alone, is toxic . Hence, we decided to explore tpm1 expression in mouse hearts by rt - pcr and sequencing . In this study, we report the expression of tpm1 in mouse heart and skeletal muscles . To the best of our knowledge, two different methods were used to quantify the expression of tpm1 and tpm1. We determined the absolute copy number as well as relative expression compared to a reference gene (18s rrna) in mouse heart and skeletal muscle . Unlike in humans, the expression level of tpm1 transcripts in mouse heart is much lower compared to tpm1. Further, there are three amino acid changes in mouse relative to human tpm1. Mouse whole heart rna was procured from stratagen and zyagen; skeletal muscle rna from biochain and zyagen . Also, we received mouse whole heart and skeletal muscle rna as a generous gift from dr . David wieczroek, university of cincinnati, cincinnati, oh . Each of these four sources of rna came from a single adult mouse . The quality of rna was determined by capillary electrophoresis using 2100 bioanalyzer, agilent technologies . The rin (rna integrity number) of the rna samples ranged from 7.2 to 9, indicating that the rnas we used were of good quality . 0.5 g of rna in a total volume of 40 l was used for preparing first strand cdna synthesis with the superscript ii (life technologies, brand island, ny) and oligodt primer following the manufacturer's specification . For each pcr amplification, nucleotide sequences for the primer pairs for amplification of various mouse genes are given in table 1 . Tpm1 contains exons 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a / b, whereas tpm1 has all the same exons except it contains exon 2a instead of exon 2b . Primer pair p1(+)/p2() was used to amplify both tpm1 and tpm1. Amplification by the primer pairs p3(+)/p2() and p4(+)/p2() will yield tpm1 and tpm1, respectively . Subsequently, an isoform - specific [32p]-labeled probe was used for southern hybridization of tpm1 or tpm1. The sequences of various primer pairs as well as probes are shown in table 1 . We amplified tpm1 and tpm1 by rt - pcr with cdna from mouse heart muscle using the mus tpm1/ primer pair . The amplified dna was run on a 1.5% agarose gel and the dna band of ~850 bp was extracted using minelute gel extraction kit of qiagen . The eluted dna was ligated to t / a cloning vector (invitrogen) following the manufacturer's specification as described earlier . The ligation mix was used for transformation of competent one shot e. coli cells (invitrogen) using the protocol supplied by invitrogen . Appropriate hybridization positive colonies were picked up from the plates after filter hybridization with [32p]-labeled probe . Exon 2a specific probe was used for tpm1 and exon 2b specific probe was used for tpm1. Colonies were grown overnight with lb medium containing appropriate concentration of antibiotic and plasmid dna was isolated using qiaprep spin miniprep kit (qiagen). Isolated plasmid dna was sequenced at the cornell university dna sequencing facility (figure 5). In order to exclude all other isoforms of the tpm1 gene, cdna was made with a negative primer from exon 9a (5-cgctctcaacgatatgactt-3), which is common to both tpm1 and tpm1 and is absent from other isoforms (figure 1). 0.5 g of rna in a total volume of 40 l was used for preparing the first strand cdna synthesis with the superscript ii (life technologies, brand island, ny) following the manufacturer's specification . Real - time quantitative rt - pcr (qrt - pcr) analysis of cdna template was performed using the lightcycler 480 real - time pcr system . Reactions were carried out in a 384-well plate using the lightcycler 480 sybr green i master kit (roche). Briefly, each well contained a total volume of 10 l, of which 2 l was cdna template and 8 l was sybr green mix (5 l 1x sybr green master mix, 2.8 l of pcr grade water, and 0.2 l of 10 m primer pair). Expression of tpm1 isoforms was determined using primer pairs for tpm1 and tpm1, (+) 5-tggaagatgagctggtgtcac-3/()5-tcaatgactttcatgcctct-3, and (+) 5-ctcgaggaggacatcgcagcg-3/()5-tcaatgactttcatgcctct-3, respectively . 18s rrna was assessed as an internal control with primer pair (+) 5-gtggagcgatttgtctggtt-3/()5-cgctga gccagtcagtgtag-3. Amplification in the absence of cdna template was also evaluated to ensure lack of signal due to primer dimerization and extension or carryover . End point melt curve analysis confirmed the presence of a single amplicon in each reaction well . A crossing threshold (ct) value was obtained, corresponding to the fractional number of amplification cycles where the pcr curve reaches a program - defined threshold amount of fluorescence . It is to be noted that qrt - pcr was performed separately with cdnas prepared from rna procured from three different sources as mentioned above . Relative quantification of qrt - pcr data was performed using the delta ct (sample ct minus 18s rrna ct) and delta delta ct (sample delta ct minus comparator delta ct) methods [15, 16]. A comparative value was calculated using the formula x, where x equals the efficiency of tpm1 or tpm1 primer pairs . This is similar to the 2 method but corrects for the assumption that the reaction is occurring with 100% efficiency . Efficiencies (e) were determined using dilution series of tpm1 and tpm1 plasmid clones with respective isoform - specific primers pairs . The lightcycler 480 software plotted the ct at each concentration against the logarithm of the fold dilution of the clone, generating a linear regression curve that calculated efficiency based on the formula e = 10 . Where tpm1 and tpm1 were compared within the same cdna sample, their efficiencies were averaged to equal 83.73% . Efficiency of 18s rrna was determined by serial dilution of mouse cdnas generated with exon 9a primer . For determination of absolute copy number, optical density was taken of mouse tpm1 and tpm1 ta clones separately using a spectrophotometer . The copy number per volume of clone in solution was determined using the equation number of copies = (ng of plasmid dna 6.02 10)/(bp length of plasmid 1 10 650), which was simplified by andrew staroscik at the uri genomics and sequencing center . 10 copies of template, which was used to create a standard curve after amplification [8, 14]. The means, standard deviations, and comparative analyses of each data set for statistical significance were done using paired student's t - test . For expression analysis, we first employed conventional rt - pcr with cdna made from total rna from mouse heart and skeletal muscle . Tpm1 rna was expressed in both mouse heart and skeletal muscle . To the best of our knowledge, this is the first report of the expression of tpm1 in mammalian skeletal muscle, albeit at a significantly lower level compared to tpm1. Figure 2 depicts the results of conventional rt - pcr analysis of tpm1 and tpm1 with cdnas synthesized from mouse heart and skeletal muscle total rna (provided by dr . David wieczroek), using a variety of generic as well as isoform specific primer pairs . Panel (a) in figure 2 represents the ethidium stained pcr products run on a 1.5% agarose gel . Lanes 1 and 2 show the pcr products amplified from heart cdna with two different primer pairs, which amplify both tpm1 and tpm1. Southern hybridization with tpm1-specific probe demonstrated stronger signals in both lanes (lanes 1 and 2, panel (b)) compared to hybridization with tpm1-specific probe (lanes 1 and 2, panel (c)). The weaker signals in lanes 1 and 2 in panel (c) compared to panel (b) indicate much lower expression level of tpm1 compared to tpm1. Lanes 3 and 4 in panel (a) represent the ethidium staining of the pcr products when amplified with tpm1 or tpm1-specific primer pairs, respectively . Southern hybridization with tpm1-specific probe shows a strong hybridization signal (lane 4, panel (b)) with the tpm1-specific primer pair . A strong hybridization signal was also detected with tpm1-specific probe (lane 3, panel (c)) with the tpm1-specific primer pair . This was further confirmed by amplification with another tpm1-specific primer pair and subsequent southern hybridization with tpm1-specific probe (lane 5, panels (a), (b), and (c)). Lanes 9 and 10 represent the pcr amplicons of mouse skeletal muscle cdna with two sets of tpm1/ primer pairs . Ethidium bromide stained gels indicate a strong expression of tpm1 in skeletal muscle (panel (a)). Hybridization signal with tpm1-specific probe (panel (b)) versus the tpm1-specific probe (panel (c)) indicates a much stronger expression of the former . However, amplification of skeletal muscle cdna with tpm1-specific primer pair (lane 11 in panel (a)) and subsequent hybridization with tpm1-specific probe (lane 11 in panel (c)) validate the expression of tpm1 in mouse skeletal muscle . Similarly, the expression of tpm1 in skeletal muscle was authenticated by the pcr amplification with tpm1-specific primer pair (lane 12, panel (a)) and by subsequent hybridization with tpm1-specific probe (lane 12, panel (b)). A much lower expression level of tpm1 compared to tpm1 in mouse heart and skeletal muscle was further substantiated by qrt - pcr as described below . Similar conventional rt - pcr results were obtained from the rna procured from stratagen and biochain (data not shown). Relative quantification of tpm1 and tpm1 in mouse heart and skeletal muscles was performed on all sources of rna . Our qrt - pcr data for copy number determination indicate that the expression of tpm1 is much lower in both heart and skeletal muscle compared to tpm1 (table 2). In relation to cardiac muscle, the data indicate that the level of expression of tpm1 transcripts is significantly higher and the level of tpm1 is significantly lower in skeletal muscle (tables 2 and 3). As the absolute copy number determination revealed that the expression level of tpm1 is significantly lower in heart and skeletal muscle, we analyzed our results by 2-(ddct) method using 18s rrna as the reference gene to determine the relative expression of tpm1 and tpm1. Melt curve data presented in figure 4(a) show different unique melting temperatures for tpm1 (left) and tpm1 (right). Also, agarose gel electrophoresis of the pcr amplified dna for tpm1 or tpm1 shows a single band for both of them (figure 4(b)) the data in table 4 validate our previous findings that expression of tpm1 is significantly lower than tpm1 in mouse heart and skeletal muscle . The fold changes of tpm1/tpm1 are significantly higher in skeletal muscle suggesting the higher expression level of tpm1 in heart as concluded from the absolute copy number data (table 2). The results support our conclusion that tpm1 expression is much higher in mouse skeletal versus heart muscle; and the level of expression of tpm1 is higher in mouse heart versus skeletal muscle (table 5). Cdna sequencing confirmed the expression of both tpm1 and tpm1 expression in mouse heart and skeletal muscle . Nucleotide and the deduced amino acid sequences of mouse tpm1 were identical with the published sequence (accession number nm_001164248.1) (data not shown). Also, the nucleotide sequences of mouse tpm1 and tpm1 as depicted in figure 5 are identical except for exon 2 . As predicted, tpm1 contains exon 2b, but tpm1 has exon 2a . The deduced amino acid sequences of mouse and human exon 2b of the tpm1 gene are identical . The deduced amino acid sequences of exon 2a of mouse tpm1 and the published exon 2a sequences in mouse tpm1 (smooth muscle tpm1 isoform) (nm_001164249.1) are also identical . However, some differences were noted when comparing the deduced amino acid sequences of mouse exon 2a with human tpm1 2a (figure 6). Amino acid residues e and a in humans are replaced by d and t in mouse tpm1, respectively . However, the most notable difference is at amino acid residue 52 . In humans, it is valine, whereas in mouse it is alanine . This amino acid residue is in the middle of the 15-mer peptide (kekllrvsederdrv) that was used as the antigen for developing antibody against human tpm1 . This alteration may cause a lower affinity of this antibody towards mouse tpm1 protein . In order to evaluate the expression of tpm1 protein, in this study, we carried out western blot analysis with isolated myofibrils from mouse heart and skeletal muscle using the human tpm1-specific antibodies . The antibodies against human protein showed several nonspecific bands with mouse heart and skeletal muscle extracts . The western results are not convincing as to whether tpm1 protein is expressed in mouse striated muscle . Both the rna and protein of this sarcomeric tm isoform is expressed in both heart and skeletal muscle in axolotl . Subsequently, we reported the expression of tpm1 transcripts and protein in human heart but not in skeletal muscle . Also, tpm1 along with tpm1 is expressed in embryonic chicken heart but not in skeletal muscle . However, the transcripts of neither tm isoform could be detected in adult chicken heart and skeletal muscle . Our present study clearly demonstrates that tpm1 rna is expressed in adult mouse heart and skeletal muscle . Unfortunately, because an anti - mouse tpm1 antibody is not available, we cannot comment on protein expression . The levels of tpm1 and tpm1 rna in axolotl heart and skeletal muscle and human heart muscle are comparable . However, the level of expression of tpm1 protein compared to tpm1 protein is very low . Hence, we cannot speculate about levels of tpm1 protein in murine striated muscles . Interestingly, the expression of tpm1 protein is increased in hearts in dilated cardiomyopathy (dcm) and heart failure human patients . However, we do not know whether this increase in tpm1 is the cause or consequence of the cardiac disease . The immunohistochemical analyses with tpm1 antibody show that it is incorporated into axolotl cardiac and skeletal myofibrils and into human cardiac myofibrils . We have shown that transfection of antisense tpm1 oligonucleotides into embryonic axolotl hearts inhibits the cardiac contractility in situ and also disarrays the cardiac myofibrils . Herein, for expression analysis of tpm1 transcripts we performed both conventional and real - time rt - pcr with total rna from mouse heart and skeletal muscle using isoform specific primer pairs . Rt - pcr data show the expression pattern of tpm1 and tpm1 in mouse heart and skeletal muscle (figure 4). We quantified the absolute copy number of tpm1 and tpm1 expressed in heart and skeletal muscle . The results presented in tables 2 and 3 show the higher copy number of tpm1 expressed in mouse heart compared to skeletal muscle . Hence, the difference in the ratio of copy numbers of tpm1: tpm1 is even more pronounced between the two tissues . The expression of tpm1 transcript is ~2- to 3-fold higher in skeletal muscle compared to heart . On the contrary, the expression of tpm1 is ~3-fold higher in heart compared to mouse skeletal muscle . The real - time pcr (rt - pcr) results using copy number procedure is also reflected from the qrt - pcr data using 2-(dct) method where we have used 18s rrna as the reference gene (table 4). We conclude that expression level of tpm1 transcripts is higher in mouse heart compared to skeletal muscle . Further, the expression level of tpm1 compared to tpm1 is significantly lower in both heart and skeletal muscle . Our analysis of qrt - pcr data by 2-(ddct) method also supports the similar conclusions (table 5). It is worth mentioning that tpm1 expressed per g of total rna is considerably higher in skeletal muscle relative to heart muscle in axolotl as well . . A higher expression level of tpm1 in skeletal muscle could be attributed to the higher requirement of tpm1 protein in skeletal muscle or due to the higher turnover of tpm1 rna or protein in skeletal muscle . An altered dynamicity of tropomyosin in heart versus skeletal muscle may contribute to higher concentration of tpm1 mrna observed in skeletal muscle . Wang et al . Compared the recovery rates of mature myofibrils in avian cardiomyocytes and skeletal muscle cells transfected with avian tpm1 or tpm1 after photobleaching and noted the marked decreased rates of recoveries of both tpm1 isoforms in skeletal muscles compared with myofibrils in cardiomyocytes . Hence, one can speculate that more transcripts of tropomyosin accumulate in the skeletal muscles cells or the transcriptional efficiency of the tpm1 gene is significantly higher in skeletal muscle cells . The deduced amino acid sequences show that three amino acid residues in exon 2a (figure 6) are different from that of human sequences . In human tpm1, the 52nd amino acid residue is valine whereas it is alanine in mouse tpm1. This alteration may contribute to a lower affinity of the tpm1 antibody [8, 12] that we used for western blot analysis to detect the expression of tpm1 protein in mouse heart and skeletal muscle . An antibody, arg1, targeted toward residues 214226 (tyr - ser - gln - lys - glu - asp - arg - tyr - glu - glu - glu - ile - lys) of human tpm1 can recognize human tpm1 but not rat tpm1. The amino acid sequence of human tpm1 differs from that of rat tpm1 by an arg - to - lys amino acid exchange in position 220 . In addition, amino acid residues e and a in humans are replaced by d and t, respectively, in mouse tpm1 (figure 6). Overexpression of human tpm1 protein in a cardiac - specific manner leads to the development of dcm in transgenic mice [12, 20]. It is to be noted that several missense mutations in exon 2b in tpm1 have been implicated in human dcm . Olson et al . Identified two mutations in exon 2b of the tpm1 gene that altered two very conserved amino acids and also reversed the charges on the surface of tropomyosin . The differences of the amino acid residues in exon 2a of human tpm1 and mouse tpm1 (figure 6) do not alter the net charges . At this juncture, we are unsure whether the absolute overexpression of human tpm1 and/or its altered exon 2a protein relative to the mouse sequence explains the dcm - like phenotype observed in transgenic mice . Obviously, one should consider creating transgenic mice with mouse tpm1 to better understand the physiological relevance of tpm1 protein.
Reduced sperm count and sperm quality are reported from many developed economies and there are also increased rates of testicular cancer manifested in western and northern europe [2, 3], australia, and northern america . It has been suggested that this negative development could be caused by increased exposure to environmental contaminants . Physical as well as chemical exposures have been associated with reduced sperm quality in association studies [510]. Chemical environmental contaminants have been shown to negatively affect reproduction and embryo development in animals [1115]. In humans, spermatozoa from infertile men demonstrate higher levels of dna damage compared to fertile men, and sperm dna damage is associated with low sperm quality [1619] and reduced fertility . Concern is being raised over the possibility that paternal germ cell dna damage in humans, induced by environmental contaminants, could have an impact on the next generation . Benzo[a]pyrene b[a]p is a carcinogenic contaminant with ubiquitous distribution and potential reprotoxic effects [2125]. B[a]p is found in coal tar, in automobile exhaust fumes (especially from diesel engines), in all smoke resulting from the combustion of organic material (including cigarette smoke), and in charbroiled food . This compound is the chemical compound whose ability to form dna adducts has been best characterized . B[a]p undergoes metabolic transformation to a diol - epoxide, bdpe, in the human organism [26, 27]. The global distribution and dna damage - inducing properties of b[a]p make it a relevant genotoxic model compound for the study of potential transgenerational effects of paternal exposure . Micrornas (mirnas), discovered in 1993, are short (1725 nucleotides) noncoding rnas which negatively regulate specific target genes by mrna degradation or translational repression . Mirnas have fundamental roles in multiple cellular processes and are also implicated in the development of multiple diseases (for a review see). Their importance is evident from phenotypes of knockout and mutant mice and from studies comparing expression profiles . Representing promising therapeutic targets and candidate biomarkers in pathophysiology, mirnas are an active area of research . Several studies implicate mirnas in the control of early embryonic development and maintenance of the pluripotent stem cell state, but the impact of environmental contaminants on mirna expression has been little studied so far . Recently, epigenetic mechanisms through which paternal influence on offspring development have received more attention, and mirnas play a key role in epigenetic regulation . Following paternal acute exposure to b[a]p four days prior to fertilization, we studied the global mirna expression profile of the developing mouse embryo . We demonstrate that genome - wide mirna expression profiling studies can be performed on a very limited number of cells and that early embryonic transcription of multiple mirnas is affected by b[a]p exposure of the fertilizing sperm . To our knowledge, this is the first report on embryonic mirna modulation, following paternal exposure to environmental contaminants . Exposed males (strain b6d2f1 from charles river laboratories, 812 weeks of age) received one i.p . Injection of b[a]p (150 mg / kg body weight) dissolved in corn oil four days prior to the ivf experiment . Timing of the exposure to b[a]p was based on pilot studies and knowledge about the most susceptible stage of spermatogenesis with respect to dominant lethal mutations . Similarly, aged control males received an equivalent volume of corn oil . At the day of the ivf experiment, cauda was surgically removed and collected in an eppendorf tube containing m2 medium (500 l, sigma). Using a pair of microscissors, a few incisions were made in the cauda and the sperm was allowed to disperse for 10 minutes in a small drop (250 l) of htf medium (embryomax, millipore) under liquid paraffin (medicult) before transfer to the ivf dishes . Experiments are based on oocytes from 36 females and sperm from 6 males (3 exposed and 3 controls) altogether . Females (strain b6d2f1 from charles river laboratories, 46 weeks of age) were injected i.p . With pregnant mare serum hormone gonadotropine (pmsg, folligon from intervet) (5 iu) three days prior to the ivf procedure . Two days later (i.e., the day before the ivf) animals received an additional i.p . Injection of human chorionic gonadotropine (hcg, ovitrelle from serono) (5 iu). Mice were killed by cervical dislocation and oviducts were collected in m2 medium (sigma). Egg clutches (1020 oocytes) embedded in cumulus cells were extracted from each oviduct . Oocytes were transferred to ivfdishes and incubated in a droplet of htf sperm containing medium under liquid paraffin for 4.5 h (37c). Oocytes from one side of the animal were combined with sperm from b[a]p exposed animals and oocytes from the other side where combined with sperm from control animals . Hence, oocytes from all animals were present in both the control group and the exposed group . After 4.5 h, the fertilized oocytes (zygotes) were washed 5x in ksom medium (embryomax millipore) before they were transferred to a drop of ksom (200 l) in a petri dish (35 mm) under liquid paraffin (medicult). The zygotes were allowed to grow for harvesting at the 2-cell, 8-cell and blastocyst stage . At harvest, 20 embryos were collected in microtubes filled with 5 l lysis medium (cellulyser, tataa) and then frozen at 80c until use . Hence, the 20 embryos contained in one sample represent a random selection of fertilized oocytes from 12 different females, all of which had been fertilized by the same male . There are no technical replicates; values are based on one pooled sample of 20 biological replicates at each stage and treatment . Embryo lysates were subjected to global mirna preamplification using sbi small rna amplification kit according to manufacture's protocol (system biosciences (sbi), mountain view, ca). The small rna amplification system consists of three steps: (1) ligation of an adapter to the 3 end of the rna, (2) reverse transcription of the rna and attachment of a 5-end adapter in the same reaction by template switching, and (3) pcr amplification of the cdna with a pcr polymerase mixture that incorporates a proof reading function . Brief description of the protocol: in the adapter ligation step rnase - free water (2 l), sbi ligase buffer (5 l), sbi 3 adaptor (0.5 l), total rna from lysed samples (5 l), and sbi ligase cocktail (0.5 l) were mixed and incubated (1 hour, 37c). In the first strand synthesis rnase - free water (4 l), sbi 3 adaptor primer (0.5 l) and ligation product from the adaptor ligation (1 l) were mixed and incubated in two steps (1 minute at 65c followed by 5 min at 42c). In the reverse transcriptase step, a mastermix consisting of sbi 5x reverse transcriptase buffer (2 l), sbi dntp mix (1 l), sbi 5 adaptor (0.5 l), sbi dithiothreitol (0.5 l), and sbi reverse transcriptase (0.5 l) were mixed with the preceding first - strand synthesis product and allowed to incubate in two steps (5 min at 42c, and then 10 min at 95c). Reactions were then kept on ice until second - strand synthesis and amplification . In the second strand synthesis and amplification step, the product from the first strand cdna synthesis step was mixed with rnase - free water (77 l), sbi 10x pcr buffer (10 l), sbi dntp mix (2 l), sbi 3adaptor primer (4 l), sbi 5adaptor primer (4 l), and sbi pcr polymerase (3 l). The reactions were transferred to a thermal cycler (eppendorf mastercycler) and underwent the following program: 5 min at 95c, 42 times (25 sec at 95c, 20 sec at 55c, 30 sec at 72c), 30 sec at 72c, and held at 15c . Following mirna amplification, genome - wide qpcr mirna expression profiling was performed using mouse mirome microrna qpcr profiler from system biosciences, which simultaneously profiles all known mouse mirnas, on an applied biosystems 7500 real - time pcr system (applied biosystems, foster city, ca). The mirna profiling was performed by qpcr using microrna - specific primers and sybr green rt2 rox mastermix (qiagen, germany). In brief, each 20 l qpcr reaction included sybr green rt2 rox master mix buffer (10 l), sbi universal reverse primer (0.10 l), globally amplified mirna samples (0.13 l), sbi quantimir microrna primer (4 l), and rnase - free water (5.75 l). The pcr program was 2 min at 50c, 10 min at 95c, 40 times (15 sec at 95c, 1 min at 60c, 35 sec at 72c (data read)), followed by a melting curve analysis step . Raw ct - values from 457 mirnas were preprocessed to remove outliers and mirnas for which there were inadequate measurements . The ct values equal to 35 cycles were considered as a limit of detection and all ct values> 35 were removed from downstream analysis . In addition, filtering criterium for missing values was set to 80%, which is the minimum percentage of existing values, and all the patterns with less than 80% existing values were removed . Only those mirnas passing quality assurance criteria were included in the downstream analysis . The raw ct - values were normalized using the mean expression value of all expressed mirnas as previously [32, 33], and the mean expression value for individual sample was calculated from mirnas with ct - values 35 cycles . The relative expression levels in samples were analyzed by the comparative ct - method [34, 35]. The fold change indicates the expression level of mirnas from paternal b[a]p exposed samples relative to that of untreated control samples . The fold change values were log2-transformed in order to make the data symmetrical around zero . Unsupervised hierarchical clustering analysis was performed to cluster variables into groups based on their similarity, and the results were visualized in a dendrogram using mev v4.7 software or j - express v2009 (molmine, bergen, norway). The mirwalk algorithm is based on a computational approach which identifies the longest consecutive complementary between an mirna and a gene sequences; mirwalk compares the gene's identified mirna binding sites with the results of eight established mirna - target prediction programs . The mirwalk database can provide mirna targets interaction information produced by eight different established mirnas prediction programs . For functional enrichment analysis of the webgestalt v2 is a web - based gene set analysis tool, and it is a suite of tools for functional enrichment analysis in various biological contexts . Webgestalt compares a user - uploaded gene list with genes in predefined functional categories to identify those categories with enriched numbers of user - uploaded genes . The webgestalt v2 enrichments analysis is based on hypergeometric statistical tests, including benjamini and hochberg multiple test adjustment . The minimum number of genes per category was set to 2 (default), and the whole mouse genome was used as reference gene set . The enrichment analysis identified the top 10 pathways with the most significant p - values . Zygotes and embryos from three preimplantation stages (2- and 8-cell as well as the blastocyst stage), fertilized either with control sperm or with sperm from b[a]p - treated mice, were used to study the effect of paternal exposure to b[a]p on the expression of a panel of 456 mouse mirnas . A total of 60 in - vitro fertilized embryos (30 derived from b[a]p exposed sperm and 30 from control sperm) at three different developmental stages were used for the final analysis . We timed our sample extraction according to 24 h cycles, with the 2-, 8-cell, and blastocyst time points referring to 24 h, 72 h, and 120 h, respectively . The stages can be readily identified up to the 8-cell stage with careful visual inspection serving as additional quality criteria upon sampling . Samples were selected exclusively from the pool of healthy looking embryos representative of that particular stage . It is well known that embryonic cell cycle duration is subject to considerable variation, not only among different embryos, but also among blastomeres within the same embryo [4042]. Figure 1 shows a venn - diagram of the number of mirnas expressed at different stages . Some differences were noted in the number of mirnas expressed at different stages among control embryos and embryos of b[a]p exposed fathers; more mirnas were expressed in the exposed group at the 2-cell and the blastocyst stage than in controls . At the 8-cell stage, there were more mirnas expressed in the control group than in the exposed group . The venn - diagram in figure 1 also reveals that more mirnas were expressed consistently across all stages among embryos of b[a]p exposed fathers compared to controls . The outcome of the quality assurance filtering criteria mentioned above was a set of 102 mirnas, and these mirnas were used in the downstream analysis . Unsupervised hierarchical clustering analysis of these 102 mirnas was performed using mev v4.7 software, and the resulting heatmap is presented in figure 2 . By visual inspection of the heatmap, we observed that the 8-cell and blastocyst embryos were clustered close to each other and shared similar expression pattern compared to the 2-cell embryo (figure 2). There were also some mirnas which were predominantly up- or downregulated following paternal b[a]p exposure in all three stages . From these dysregulated mirnas, we selected six mirnas from the upregulated part and six from the downregulated part of the spectrum for target - genes analysis (figure 2). We were primarily interested in b[a]p - induced effects, and therefore we consistently selected the up- or downregulated mirnas across all stages . We then used the mirwalk database in order to identify potential mirna targets for the selected b[a]p dysregulated mirnas . The intersection of identified target genes from at least five prediction programs was chosen, and the numbers of predicted target genes for each selected mirna are shown in table 1 . Each mirna can regulate numerous target genes and therefore has the potential to alter multiple biochemical pathways . To investigate what pathways may be regulated by our twelve selected dysregulated mirnas, we evaluated the biological functions of their predicted target genes using webgestalt v2 . We correlated predicted target genes with the kegg (kyoto encyclopedia of genes and genomes) biochemical pathways in order to identify enriched pathways . The results from enrichment analysis represent a global picture of pathways that are significantly enriched with target genes for dysregulated mirnas following paternal b[a]p exposure . Significantly enriched kegg pathways annotating the predicted target genes for the twelve dysregulated mirnas following paternal b[a]p exposure are presented in table 2 . The complete set of significantly enriched pathways annotating target genes are listed in supplementary table 1 (see supplementary material available at doi:10.1155/2012/407431). Examples of some significantly enriched pathways are cytokine - cytokine receptor interaction, insulin signaling pathway, regulation of actin cytoskeleton, apoptosis, and mapk signaling pathway; these are shown in table 2 . The identified pathways are enriched mainly with target gene sets for the six upregulated mirnas following paternal b[a]p exposure . Kegg pathways significantly enriched in target gene sets for the six downregulated mirnas are involved in cell cycle, neuroactive ligand - receptor interaction, tgf - beta signaling pathway, calcium signaling pathway and chemokine signaling pathway (table 2). We wanted to investigate how the expression pattern of our target genes are modulated in other reported systems in which b[a]p - induced gene expression modulations has been studied . To this end, we performed a search in the publicly available gene expression databases, such as ncbi gene expression omnibus (geo) and arrayexpress . Here, we wanted to identify genes from our target gene list that have been modulated by b[a]p exposure to male mice in other systems and at the same time see if the expression pattern of these genes anticorrelate with the expression pattern of the twelve dysregulated mirnas in our study . Since mrna expression profiling data from experiments similar to our study are not available, we used the microarray gene expression data from verhofstad and coworkers, represensting a study of gene expression analysis in mice testis following one acute b[a]p exposure (13 mg / kg bw) by oral gavage . Spermatogenesis takes place in testis, and comparing gene expression patterns between the two types of experiments might be useful . Verhofstad and coworkers studied b[a]p exposure - induced changes in gene expression patterns in testis in both wild - type and xpc - knockout mice . In order to compare our target gene list with the testis gene expression microarray data, we downloaded the raw microarray data (geo accession number: gse17979) deposited in the geo database . These microarray data (the gpr - file: genepix results file) were reanalyzed as described previously; all data processing was performed in j - express v2009 . Only the microarray data from wild - type mice exposed with b[a]p and untreated controls were used for reanalysis . The log2-transformed ratios of the processed intensities were further analyzed by sam (significance analysis of microarray). Sam analysis was conducted in order to identify genes whose mean expression level is significantly different between b[a]p exposed and untreated control samples . This resulted in 345 genes identified as significantly differentially expressed (fdr <10%). We then conducted similarity searches between our target gene list and sam - identified gene list; this process identified 63 genes which showed anticorrelated expression pattern to our dysregulated twelve mirnas . Of those genes, 44 were downregulated and 19 were upregulated in mice testis following b[a]p - exposure . Figure 3 shows hierarchical clustering analysis of these 63 genes . By visual inspection of the heatmap (figure 3), we observed that b[a]p - exposed samples clustered close to each other in one branch while untreated control samples clustered in the other branch . The gene expression profiles constituted by these altered genes along with their anticorrelated mirna can be used as a basis for identification of b[a]p - exposure gene expression signatures . Using the results of both mirna and mrna expression profiling in combination with what is known about mirna functions we expect that identification of anticorrelated mirna - mrna pairs will narrow down the target gene list and refine the predicted mirna - mrna interactions . It appears, based on the mirz database, that some of the twelve dysregulated mirnas, most notably mir-210, mir-294, and mirs-466f-5p are highly expressed in several tissues, whereas others (mir-139 - 3p, mir-197, mir-1896, mir-346, and mir-1906) have not been detected . The most important finding from this work is that multiple mirnas are differently expressed in embryos of b[a]p exposed fathers relative to control embryos . The functional implications of early xenobiotic induced suppressed or enhanced single mirna expression profiles in developing embryos are not well understood; however, given the broad array of gene transcripts targeted by mirnas, the perturbations are likely, at some level, to be detrimental to the development of the embryo . The highly dynamic physiology of the developing embryo is reflected in dynamic mirna expression profiles . Maternally inherited mirnas are abundant in the mouse zygote, but many of the maternal gene products are quickly downregulated during the maternal - zygotic transition; some are downregulated by as much as 95% between the 1- and 2-cell stages . A 60% decrease in mirna expression levels has been reported, indicating that mirnas may be degraded similar to maternal mrnas . Transcription of zygotic rnas begins at the 2-cell stage, and ~2.2-fold increases in mirna expression have been noted from the 2- to 4-cell stage . In the cluster analysis (figure 2), the 8-cell stage and the blastocyst stage cluster together, indicating that the 2-cell stage exhibited a somewhat different expression profile from the two other stages . The predicted target genes for mirnas that were found to be dysregulated in the present study by paternal b[a]p exposure participate in numerous biochemical pathways . Among the enriched kegg pathways were those related to metabolism, cancer, cell cycle, apoptosis, mapk signaling, cytokine - cytokine receptor interaction, and tgf - beta signaling (table 2 and supplementary table 1). The predicted genes affected by the b[a]p - induced mirna aberrations overlap with previously published b[a]p - sensitive testis gene dataset; the resulting genes are shown in figure 3 . We did not have any information available on embryonic expression of mirna target genes in our current study . The testis dataset from verhofstad represents the closest match we could find in the literature, but it is not known to what extent gene expression in the b[a]p exposed developing embryos would match the testis gene expression of b[a]p exposed mice; we are pursuing this question . Perhaps the most interesting mirna for embryonic development is mmu - mir-294, a member of the mir-290 cluster . In mouse embryonic stem cells (es) mir-294 has been shown to promote pluripotency by regulating a subset of c - myc target genes and upregulating pluripotency - associated genes such as lin28 . It has been estimated that the mir-290 cluster alone makes up greater than 70% of the total quantity of mirnas in es . Expression of this cluster is rapidly downregulated upon differentiation, coincident with an elongation of the cell cycle . The mir-290 cluster maintains a very short cell cycle in es by suppressing the g1/s restriction . A similar suppressed g1/s restriction is observed in cancer cells, and it is interesting to note that mir-106b promotes cell cycle progression in a breast cancer cell line, by mechanisms similar to the mir-290 cluster in es, indicating similarities in the molecular control of the cell cycle of embryonic and cancer cells . In our analysis, the predicted target genes for mmu - mir-294 were enriched in pathways related to mapk - signaling, apoptosis, and cancer . A recent report found that the mirnas-290 cluster was more abundantly expressed in the inner cell mass than in the trophectoderm of the blastocyst, and the authors suggest that this asymmetric pattern of stemness - controlling mirna expression contributes to cell specialization . Another gene found to be downregulated in developing embryos following paternal b[a]p exposure was mmu - mir-210 . Controlling of the mitochondrial metabolism during hypoxia has been shown to involve mir-210, which is readily and dynamically affected by hypoxia inducible factor 1 (hif-1), the master regulator of the hypoxic response . The cell cycle regulator e2f3, the receptor tyrosine kinase ligand ephrin a3, and the dna repair protein rad52 have all been studied as targets for repression by mir-210 . The iron - sulfur cluster assembly proteins iscu1 and iscu2 have been identified as direct targets for mir-210 in human lung endothelial cells . Besides mitochondrial metabolism, the mir-210 may also be involved in other hypoxia - dependent processes such as iron metabolism, which is controlled by iscu1/2 activity . Furthermore, hypoxic repression of mitochondrial function by mir-210 and iscu1/2 could theoretically trigger mitophagy a form of autophagy in wihich defective mitochondria mitophagy has been characterized as a - hif - dependent mechanisms, but it remains to see if mir-210 itself induces mitophagy . Among the genes that were downregulated, the most dramatic response was seen for mmu - mir-483 . Aberrant expression of mir-483 has been reported in mice fed high - fat diets indicating a role of this mirna in energy metabolism . Consistent with the identification of mir-483 - 5p as an intronic mirna encoded within the second intron of the igf2 gene, a recent report confirms coexpression of mir-483 - 5p and insulin - like growth - factor 2 (igf2). Igf2 has been primarily implicated in the regulation of prenatal growth, but mice carrying the igf2 transgene have also been shown to have reduced bodyfat, and reduced igf2 expression in adults have been shown to be accompanied by increased fat deposition . In their report, ma and coworkers found that overexpression of mir-483 - 5p lead to a decrease rather than an increase in igf2 mrna and the authors suggest that mir-483 - 5p regulates igf2 expression both by suppressing socs3 and via an additional undiscovered mechanism . Another mirna that was found to be downregulated in embryos of b[a]p exposed fathers relative to embryos of control fathers was mir-346 . Mir-346 has been shown to be involved in regulation of carcinogenesis, inflammatory response, and differentiation [7173]. Interestingly mir-346 has also been shown to target receptor - interacting protein 140 (rip140) mrna, and thereby upregulate its protein expression without altering mrna levels . Rip140 is a transcription coregulator that modulates the activities of many nuclear receptors and transcription factors . The physiological function of rip140 has been demonstrated in a gene knockout animal model and cell - line based systems; these include roles in gluoconeogenesis, glycolysis, fatty acid oxidation, and mitochondria biogenesis as well as modulation of hormone target genes [7679]. The reduced expression of mir-346 observed in embryos of exposed fathers in the present study could indicate reduced rip140 transrepression of gene regulation . A conservative evaluation of up- and downregulated mirnas in developing embryos is advisable due to the inherent complexity of these mechanisms . Still, the study of gene and mirna expression profiles in developing embryos descending from exposed fathers, as exemplified in this report, could open up new avenues in our understanding of sublethal transgenerational effects of environmental exposure . Previous experiments looking at mirna expression in developing embryos have pooled hundreds of mouse embryos; here we demonstrate that mirna expression profiles can be obtained from very few embryos . This illustrates a potential of similar experimental approaches in relation to the reach eu directive (registration, evaluation, authorization and restriction of chemicals), implying that chemicals may be tested for effects on fertility and development using very few animals . Furthermore, mrna expression profiling of the target genes from in - vitro fertilized embryos of b[a]p exposed fathers would have strengthened the findings . There may also be some incidences of false predictions, among the identified target genes . To reduce the likelihood of such errors, we narrowed and refined the list of target genes by comparing them with publically available gene expression data . In conclusion, preconceptional paternal exposure to b[a]p may affect mirna expression in the developing mouse embryo . More research is needed to fully appreciate the implications of early dysregulated mirna expression, but given the wide array of cellular processes targeted by mirnas, undesired consequences are expected . Gene and mirnas expression in early embryos may provide valuable knowledge about potential transgenerational effect of sublethal exposure to exogenous compounds.
Nowadays changing the position of inserted implants is done in individual clinical experiments . In many cases the integrated implants are not in an appropriate position and can cause pressure to the dental nerves, maxillary sinus membrane damage or many difficulties in prosthetic procedure due to incorrect implant direction . On the other hand the implant may rotate during the abutment screw tightening or prosthetic treatment in immediate, early or delayed loading.1 during these procedures a sheer force is transmitted to implant - bone interface . This strength obviously has a detrimental effect on the bone next to the implant and could lead to disintegration.2,3 rotation of an implant after eight weeks of healing, based on contractual implant success benchmark, could be a sign of failure and in this situation implant removal would be planned even in the absence of the other established indicators of non - osteointegration.4,5,6,7,8 in other words loose implants are considered as failure and are prescribed to be removed.1 but several studies have shown that loose implant can result from the absence of bone - implant contacts,1 and is not considered as the failed implant.1,9,10,11 there is a difference between rotational mobility and buccolingual mobility.2 according to observations from clinicians, despite the possibility of non integration implicated by the rotation,4 it seems acceptable if the implant is allowed to heal for an additional period of time, integration of an implant can be re - established.1,12 considering the resistance to 20 ncm reverse torque is a parameter for osseointegration and a measure of functional stability,13 so clinicians have to decide how long they should wait for re - integration of the implant or when they should remove it from the mouth.2 however, there are very limited data supporting their decision at present.2 there is no basic and fulfilling study in this field therefore more research due to the increasing use of implant in the world and also the interest in early and immediate loading of implants is needed.2,4 this investigation is designed to find the answer to two questions: first, the best time and the success of counter torquing or transposition of inserted implants, second: whether it is possible, for any given reason, to transport implants in the bone once they are integrated . The protocol of this study was approved by the ethics committee of shahid beheshti university school of dentistry, tehran, iran, and the national animal care society, tehran, iran . A period of 10 days was considered to standardize the diet and environmental conditions of the dogs.14 all the procedures were done by one clinician . An overall number of thirty implants (implantium, republic of korea, 3.80 mm 10 mm) were used in this study, that is, three in each dog's tibia making it six for each individual dog . The consensus on dividing implants into six groups was made: 1) one week control (1wc), 2) eight week control (8wc), 3) one week counter torque (1wct), 4) one week transposition (1wt), 5) eight week counter torque (8wct); and 6) eight week transposition (8wt). The dogs were in full non - diet regime before operation with no water for four hours . An intramuscular (i m) injection of ketamine 10%, (10 mg / kg) (rotexmedica co, gmbh, germany) and xylazine 2%, (1 mg / kg) (behyar saman pharmaceutical co, tehran, iran), was used to sedate the animals prior to operation . The iv injection sodium thiopental 5% (behestan darou co, tehran, iran) with average dose (10 mg) was used for general anesthesia . The tibia was rinsed thoroughly with saline (shahid ghazi pharmaceutical co, tabriz, iran) and povidine - iodine (tolid darou co, tehran, iran). Following operation, animals were taken care of according to the protocol of tehran veterinary school, tehran, iran, with a special diet and supporting medicament . Prophylactic i m 6.3.3 penicillin (jaber ebne hayan laboratories, tehran, iran), dexamethasone 2 cc (darou pakhsh, tehran, iran), b complex 2 cc (exire pharmaceutical co, tehran, iran), mefenamic capsule 250 mg (razak laboratories, tehran, iran) per 12 hours and tramadol tablet 100 mg (razak laboratories, tehran, iran) per 12 hours was administered daily the surgical implantation was performed through a crestal incision4 and the tibia metaphyses were exposed by full thickness flap and with the ample reflection of the periosteal flap.4 one implant was inserted in the proximal part of each tibia (two implants in each dog) during vigorous irrigation with sterile saline.1 the implants were inserted 1 mm subcrestal in bone (fig . After one week, in second session, all animals were anaesthetized for the second time, as described above.1 randomly, one implant in one tibia of each dog was counter torqued by applying a reverse force with a ratchet and the implant in another tibia was transposed . Also, three new implants were inserted in each dog so that the two implants in two different tibiae in each dog were considered as the eight - week group and another one was considered as the first control group (fig . After eight weeks, during the third session, the one week group implants and first control group implants were counter torqued using mark-10 universal torque series sensor stw and the force values were read using a mark-10 force / torque indicator model bgi (jlw instruments, chicago, il, usa).4 the torque sensor was fitted with a special extension attached to the implant mount for access,4 once connected, the torque indicator system was placed in peak - force ready mode,4 and the increasing counter torque was applied until the detachment from bone was observed,4 then peak value was measured,4 and the display on the indicator was recorded.4 the implant was then pro - rotated back in to its original position . Afterwards, one implant which was not manipulated was counter torqued and another one was transposed in each dog (fig . 2a, fig . Thus, one new implant (in the tibia that had only two implant) was inserted in each dog that would be assumed as the second control group (fig . After another eight weeks, during the fourth session, the eight - week group and second control group implants were counter torqued to lose by applying stw and bgi which recorded the peak force required to detach the implant . The implants were then returned to their pre counter torque position (fig . 1, fig . 2d and fig . Thus, the groups were compared from the view of integration (ncm) with complete randomized block anova . They were assigned to six groups for study: 1) first control; 2) second control; 3) 1 wct; 4) 1wt; 5) 8 wct; and 6) 8 wt . Two of the implants in dog #1 of the 8 wct and 8 wt groups could not be liberated because the hexes of the implants were stripped . Therefore they were excluded and the second control in dog #1 was excluded accordingly . During the third and fourth sessions of surgery a very thick bone was formed above the fixtures, they were carefully removed with trephine drills so that the implants were not injured . The data of total 6 groups confirmed the normality with using one - sample kolmogorov with minimum eventuality of 0.796 . Data variance equality was studied and assigned to three groups related to 1 week (first control - 1 wct - 1 wt) with eventually 0.288 and in three groups related to eight week (second control - 8 wct - 8 wt). For comparing the quantity of osseointegration (ncm), completed randomized block anova in three groups (first control - 1 wct - 1 wt) was used . However the mean quantity of osseointegration revealed differences in the three groups (first control - 1 wct - 1 wt) but they were not significant (table 1). 3 . In order to compare the quantity of osseointegration (ncm), completed randomized block anova in three groups (second control - 8 wct - 8 wt) was used . But the mean quantity of osseointegration displayed differences in the three groups (second control - 8 wct - 8 wt) but they were not significant (table 2). 4 . In this manner, no significant difference was discovered between the two study groups (first control - 1 wct) with p=.90, two research groups (first control - 1 wt) with p=.116, two study groups (second control - 8 wct) with p=.145, two research groups (second control - 8 wt) with p=.536, two research groups (1 wct - 8 wct) with p=.234, two study groups (1 wt - 8 wt) with p=.592 . The increased demand for early and immediate loading treatment plan in patients with implant supported dentures has led the clinicians to unscrew the implant's cover screw earlier . However, this eventuality could be expected because of not having adequate time for osseointegration.1,2,4 bone healing around the implant depends on different factors . The stem cells which migrate there are only transformed into fibroblast as a result of micro movements then consequently fibrointegration occurs . So a parameter of assessing the attainment of osseointegration is a resistance to minimal 20 ncm reverse torque for preventing micromovement.4,12 many clinicians have tacitly accepted the counter torque test as a clinical indicator of successful integration.4,12,15 currently, researches emphasize the formation of a strong bed for implant by a' second callus' . Establishing and superimposing of this callus to the primary healing response will produce a stronger implant bed.4 another implication could be when implants were not inserted in an appropriate place or in close proximity to anatomic area such as maxillary sinus, inferior alveolar nerve and mental foramen . In these cases which force the implants to exit, the evaluation of the effect of utilizing counter torque is required.4,16 the present study is solely aimed at the clinical assessment of the resistance of implant following counter torque test in two intervals of one week and eight weeks . The results of this study are supported by the findings in previous reports . In our study, reintegration is possible after counter torque test is carried - out on integrated implants . This outcome was achieved after eight weeks as healing time in eight - week rotation groups . The results of the present study also confirm the findings of moriya et al.2 while the counter torque is closer to implantation time, the result is more preferable and even higher than the control group . The samples of the lucente's research were 11 humans therefore the number of the implants was statistically lower than this study . The present study showed that implant reintegration is possible after counter torque test (physical dislocation of the implant from the healing bone) in integrated implant.4 the result refers to the eight - week rotation groups . This is in accordance with findings of ivanoff et al.1 and lucente et al . In the animal study of morberg and albrektsson,17 who used rotational strain to the implant to break osseointegration, subsequent integration was achieved.2 several studies have reported good prognosis of rotated implants finally functioned without any clinical difference from non - rotated implants.2,18,19,20 transposition of the implant have been reported only in rare situations when the implant has been transposed with its surrounding bone to correct the implant position.21 however in the present study, for the first time, implant transposition has been carried out without surrounded bone and, with / without detachment, the implant was separated from the adjacent bone and installed in the other cavity . A number of them were removed one week and the others eight weeks after the implantation . The evaluation of the implants after eight weeks healing process revealed that the osseointegration has occurred . The mean value of the 1wt group (145/20 ncm) has been increased in comparison with the control group (109/40 ncm). On the other hand, the mean value of the 8 wt group (152 ncm) has decreased in comparison with the control group (182/60). This is considered a suitable osseointegration while the implant surface is covered with some osseous particles and its uncontaminated transposition from one cavity to another will cause osseointegration . Following the implantation, the clot is formed around the implant . This clot will then be organized after two days and the fibroendothelial cells and angiogenesis are observed . Also, because of different growth factors released, especially tgf (transforming growth factor), there will be a proper condition for migrating and differentiating of cells that make up osseous matrix . An important factor in the first week is the high number of stem cells and osseous callus that are highly elastic . Also, due to the presence of stem cells, osteoid will be secreted and the calcification will take place . Therefore after the counter torque or transposition, the osseointegration will be achieved, proceeding the repair / healing phase . Is that the implant surface is covered with gf (growth factor), stem cells, young connective tissue and primary osteoid . After eight weeks, the woven bone is formed at the site, the elastic callus is not present at this time, and if the counter torque or transposition is applied, due to the secondary pressure site, the resorption is also probable.22 even though following bone detachment some bone particles remain on the implant surface and cause new sites of early junctions . Based on the frost theory of the regional acceleratory phenomenon (rap),1,23,24,25 which is one aspect of healing after operation, a noxious stimulus speeds up bone modeling and remodeling.1 therefore one can hypothesize that bone detachment from the implant as a result of counter torque or transposition leads to the activation of the cytokines (e.g., tgf1, aff, bff and bmp2,7), which is the same mechanism as in any other traumatic event in the bone.4,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41 according to the mechanism of the action described by davies,4,16 it seems that the areas that did not have adherent bone were likely to be saturated with blood released from disrupted local vessels after the counter torque or transposition.4 in other words the reintegration success in these implants may be a function of the cellular processes in which the surfaces attach and secure the blood clot,4 anchoring fibrin that provides a pathway for the osteoblasts to migrate to the surface of the implant and initiate de novo bone formation.4 therefore, at final evaluation, the counter torque force also had to overcome the resistance provided by newly formed interfacial bone.4,16,42 this research showed that if the implant was counter torqued or transposed after one or eight weeks following implantation, osseointegration was achieved after the healing period . The conclusions of this study are those that follow: after the counter torque process, one week or eight weeks following the implantation, osseointegration was achieved . After transposing the implant, one week or eight weeks following the implantation, osseointegration was achieved . Comparing the one week and eight week intervals, the one week would result in a stronger osseointegration, when comparing to the values with the control group . Comparing the one week and eight week intervals, the implant transposing after one week is suggested due to its better results.
The introduction of martensitic shape memory (sm) nickel - titanium (niti) alloy in the manufacturing of rotary endodontic instruments has led to relative increase in their flexibility, cutting efficiency and cyclic fatigue resistance . These alloys were popularly termed as m - wire, cm - wire (controlled memory) and thermal memory wire by different manufacturers . The unique nature of the martensitic sm alloy is the presence of stable martensite phase at body temperature . As the austenite is relatively more rigid, they remain in the deformed state even after the withdrawal of stress due to the absence of conventional stress - induced martensitic transformation as observed in superelastic (se) niti alloys . Although, deformed martensitic niti instruments recover their shape when heated during sterilization, few instruments undergo permanent plastic deformation making them destined for single use . Also, separation of rotary instruments during instrumentation without warning is a major hindrance for the success of root canal treatment . Therefore, a supplementary treatment that increases the martensite content of sm alloy would be ideal to overcome the drawbacks and improve the clinical performance of these instruments . Cryogenic treatment (ct) is a supplementary procedure of exposing metals to very low temperatures, which affects the entire cross - section, unlike purely surface treatment . Based on the soaking temperature, the process may be deep ct (dct) or shallow ct (sct). Dct of se niti endodontic instruments has shown improved cutting efficiency and microhardness . The two main mechanisms responsible for the improvement in steel tool properties following ct are a complete transformation of retained austenite to the martensite phase and precipitation of micro - carbide particles . Despite extensive reports on the enhancement of mechanical properties of steel tools following ct, their impact on the microstructure of sm niti alloys is not studied so far in the literature . Therefore, the aim of this preliminary study was to investigate the role of dry ct conditions on the microstructure of martensitic sm niti alloy . Experiments were conducted on ni-50.8 wt% ti sm alloy with austenite finish temperature (af) of 46c in the form of sheet (2.2 mm thickness) and rod (9 mm diameter) of same ingot which complies with the specifications of american society for testing materials (astm) f2063 - 05 . Five cylindrical specimens (10 mm height; 9.0 mm diameter) and five sheet specimens (15 mm 15 mm square) were sectioned from the as - received niti sheet and rod with the help of wire electrical discharge machine (aq300 l; sodick, shaumburg, il). The protocol for dry ct and the experimental setup has been followed earlier in the previous study . The specimens were randomly divided into four experimental groups based on the soaking temperature and time with a control group as follows: dct 24 group: 185c; 24 h, dct 6 group: 185c; 6 h, sct 24 group: 80c, 24 h, sct 6 group: 80c, 6 h and control (ctrl) group: no treatment . The constant cooling rate and warming rate used during dry ct was 1c / min (3 h) and 0.6c / min (6 h) respectively . The cross - sections of five niti cylindrical specimens (one specimens from each group) were polished with a standard sequence of abrasives (carbimet paper discs, buehler: 240 and 600 grit) and alumina paste (alpha micropolish, buehler: 6 m, 1 m and 0.5 m particle sizes). The polished surfaces were etched using an etching solution of composition hf + hno3 + h2o with a volume ratio of 1:4:5 by swabbing technique . The microstructures of the specimens were examined with an optical microscope (leica dm4000 m led; leica microsystems cms gmbh, germany) and an scanning electron microscope (sem) (cx-200; coxem ltd ., daejeon, south korea). Forty - five lines per mm were used for the calculation in both vertical and horizontal direction . The elemental composition of different zones namely, the matrix, grain boundaries, martensitic variants and precipitates was determined for the sm alloy using scanning electron microscope (cx-200; coxem ltd ., daejeon, south korea) coupled to energy dispersive x - ray spectrometer (sem - eds). The quantitative analysis was performed in nonstandard analysis mode employing zaf correction methods using the software . X - ray diffraction (xrd) study was performed on polished sheet specimen from each group at room temperature using x - ray diffractometer (miniflex ii - c, rigaku, tokyo, japan). The xrd system was operated under the following conditions: cu - k rotating anode (= 1.5406), 40 kv accelerating voltage, 40 ma beam current, 10-100 2-theta range at 0.02 steps and 2 s acquisition time per step . A silicon standard was used for calibration of the diffractometer (640b silicon powder xrd spacing, standard reference material; nist, gaithersburg, md, usa). The (hkl) planes corresponding to the peaks of austenite, martensite and precipitates were identified using the icdd database (pdf release 2004; international centre for diffraction data, newton square, pa, usa). The protocol for dry ct and the experimental setup has been followed earlier in the previous study . The specimens were randomly divided into four experimental groups based on the soaking temperature and time with a control group as follows: dct 24 group: 185c; 24 h, dct 6 group: 185c; 6 h, sct 24 group: 80c, 24 h, sct 6 group: 80c, 6 h and control (ctrl) group: no treatment . The constant cooling rate and warming rate used during dry ct was 1c / min (3 h) and 0.6c / min (6 h) respectively . The cross - sections of five niti cylindrical specimens (one specimens from each group) were polished with a standard sequence of abrasives (carbimet paper discs, buehler: 240 and 600 grit) and alumina paste (alpha micropolish, buehler: 6 m, 1 m and 0.5 m particle sizes). The polished surfaces were etched using an etching solution of composition hf + hno3 + h2o with a volume ratio of 1:4:5 by swabbing technique . The microstructures of the specimens were examined with an optical microscope (leica dm4000 m led; leica microsystems cms gmbh, germany) and an scanning electron microscope (sem) (cx-200; coxem ltd ., daejeon, south korea). Forty - five lines per mm were used for the calculation in both vertical and horizontal direction . The elemental composition of different zones namely, the matrix, grain boundaries, martensitic variants and precipitates was determined for the sm alloy using scanning electron microscope (cx-200; coxem ltd ., daejeon, south korea) coupled to energy dispersive x - ray spectrometer (sem - eds). The quantitative analysis was performed in nonstandard analysis mode employing zaf correction methods using the software . X - ray diffraction (xrd) study was performed on polished sheet specimen from each group at room temperature using x - ray diffractometer (miniflex ii - c, rigaku, tokyo, japan). The xrd system was operated under the following conditions: cu - k rotating anode (= 1.5406), 40 kv accelerating voltage, 40 ma beam current, 10-100 2-theta range at 0.02 steps and 2 s acquisition time per step . A silicon standard was used for calibration of the diffractometer (640b silicon powder xrd spacing, standard reference material; nist, gaithersburg, md, usa). The (hkl) planes corresponding to the peaks of austenite, martensite and precipitates were identified using the icdd database (pdf release 2004; international centre for diffraction data, newton square, pa, usa). Optical microscopic images [figure 1a] of all etched specimens showed equiaxed grains with well - defined boundaries . The average diameter of the grains was approximately 25 m with astm grain size number of 7.7 in all the groups . Table 1 shows the volume of martensite that is observed in the images of different groups . (a) optical micrograph of shape memory nickeltitanium (niti) alloy (black circle denotes the martensite variant of the grain), (b) scanning electron microscope micrograph (white circle denotes the martensite variant); holes (black arrow) and precipitates (white arrow) in the niti matrix are present, (c) eds spectrum showing the elemental composition comparison of martensite and austenite phase in all the groups the martensite variants were well appreciated under higher magnification with the sem images [figure 1b]. The eds spectrum reveals the elemental composition of the niti specimen, which is nickel - rich [figure 1c]. The average elemental composition of the as - received sm alloy was ni 50.8 wt% and ti 48.4 wt% with trace elements [table 2]. The regional variations in the composition revealed an increase in ni content, especially in the precipitates and twinned regions . Elemental composition of sm niti alloy at different regions x - ray diffraction pattern [figure 2] of the specimens at room temperature contained the following peaks: (110), (200) and (211) atomic planes for austenite; (11), (002) and (012) atomic planes for martensite; (203), (205) and (220) atomic planes for tini3 precipitates; (220) and (042) atomic planes for ti2 ni3 and ti3ni4 precipitates respectively . Average intensities and full width at half - maximum height of the peaks corresponding to austenite (211) and martensite (002) were used to describe the qualitative change in crystal size and content of the sm niti alloy [table 1]. X - ray diffraction patterns for the shape memory nickel - titanium alloy at room temperature . (a) all the groups . Optical microscopic images [figure 1a] of all etched specimens showed equiaxed grains with well - defined boundaries . The average diameter of the grains was approximately 25 m with astm grain size number of 7.7 in all the groups . Table 1 shows the volume of martensite that is observed in the images of different groups . (a) optical micrograph of shape memory nickeltitanium (niti) alloy (black circle denotes the martensite variant of the grain), (b) scanning electron microscope micrograph (white circle denotes the martensite variant); holes (black arrow) and precipitates (white arrow) in the niti matrix are present, (c) eds spectrum showing the elemental composition comparison of martensite and austenite phase in all the groups the martensite variants were well appreciated under higher magnification with the sem images [figure 1b]. The eds spectrum reveals the elemental composition of the niti specimen, which is nickel - rich [figure 1c]. The average elemental composition of the as - received sm alloy was ni 50.8 wt% and ti 48.4 wt% with trace elements [table 2]. The regional variations in the composition revealed an increase in ni content, especially in the precipitates and twinned regions . X - ray diffraction pattern [figure 2] of the specimens at room temperature contained the following peaks: (110), (200) and (211) atomic planes for austenite; (11), (002) and (012) atomic planes for martensite; (203), (205) and (220) atomic planes for tini3 precipitates; (220) and (042) atomic planes for ti2 ni3 and ti3ni4 precipitates respectively . Average intensities and full width at half - maximum height of the peaks corresponding to austenite (211) and martensite (002) were used to describe the qualitative change in crystal size and content of the sm niti alloy [table 1]. X - ray diffraction patterns for the shape memory nickel - titanium alloy at room temperature . (a) all the groups . Cryogenic treatment of sm niti alloys for endodontic use appears to be a promising field of research due to the expected transformation of retained austenite to martensite as observed in stainless steel alloys . The sm alloy regains its shape on heating due to the martensite - austenite transformation, which make the file se before cooling . Deep cryogenic processing uses temperatures around 185c because this is a minimum temperature easily obtainable with liquid nitrogen . The purposes of selecting raw niti specimens in this study are as follows: the intention was to assess the effect of ct alone.the influence of thermomechanical processing and manufacturing methods (machining / twisting) were eliminated.the dimension of the specimens were customized according to the requirement.the complicated geometry of the endodontic files was avoided . The influence of thermomechanical processing and manufacturing methods (machining / twisting) were eliminated . The complicated geometry of the endodontic files was avoided . The structural analysis of the surface and near - surface (50 m) the grain size of the niti specimens obtained from optical micrographs did not show major variations between the groups . Martensitic variants were observed in specimens of all the groups including the ctrl . This could be attributed to the af temperature of the sm niti alloy which is above the room temperature . The volume of martensite obtained from micrographs in dct 24 group was relative higher (4.1%). In our previous study, specimens treated with dct 24 conditions had showed a significant reduction in vickers hardness and reciprocatory sliding wear resistance of sm niti alloy (unpublished observations). The conversion of retained austenite into martensite with a substantial increase in the volume of martensite phase could be the reason for such peculiar behavior of the alloy . Although the difference in volume of martensite at a given site among the experimental groups was minimal, they do not actually reflect the exact volume of the entire cross - section of the alloy . In our previous experiments, dsc analysis on the as - received sm niti alloy used in this study showed two - stage (austenite r - phase martensite) phase transformation during cooling (unpublished observations), which is similar to the transformation behavior of sm niti instruments . Ti3ni4 precipitates observed in the micrographs influence the martensitic transformation behavior of the alloy by favoring the formation of r - phase and altering the ni content of the matrix . The ni content in the matrix is reduced due to the presence of ni - rich intermetallic phases, which subsequently results in the increase of af temperature and overall martensite phase transformation temperature range . The xrd spectra corresponding to 2 range of between 35 and 80 is of special interest as most of the austenite and martensite peaks were located . The peak intensity of martensite (002) phase and the additional martensite peaks (11 and 012) was relatively high in the xrd pattern of dct 24 specimen [figure 2 and table 1]. This could be attributed to a prominent increase in the martensite volume of the specimen as observed from the micrograph . Crystallites and subsequently the grains in the as - received niti specimens preferentially grow along the martensite (002) lattice plane following ct . The (110) peak of the austenite phase was not observed in both control and experimental groups except dct 24 . This could be due to overlapping of (110) peak with the prominent (11) martensite peak . The presence of martensite phase at room temperature in the niti specimens could be attributed to the composition and heat treatment of the alloy . In dct 24 specimen, the crystallite size and peak intensity of austenite was reduced, whereas they are increased for martensite . Although minor variations in values were observed in other experimental groups, they cannot be attributed to the variations in the ct parameters . It is known that the fatigue resistance and flexibility of the sm rotary instruments is remarkably increased due to the presence of proportionately higher martensite phase . Therefore, the life of the rotary endodontic instrument made out of our new experimental alloy will be extended in a great way . Furthermore, soaking period plays an important role in altering the microstructure of sm niti alloy as far as dct is concerned . Longer soaking time of 24 h provides adequate time for the transformation of retained austenite to the martensite phase . Under the limitations of this study, deep dry ct with 24 h soaking period increases the martensite content of sm niti alloy without altering the grain size . By correlating our previous experiments and the current findings, deep dry ct with 24 h soaking period could be recommended as a supplementary method in the manufacturing in order to extend the life of sm niti endodontic instruments . Further investigation is required to evaluate the effect of ct on the fatigue properties of the bulk specimens.
An estimated 10 million us adults have peripheral artery disease (pad), a manifestation of systemic atherosclerosis that has been linked to reduced functional capacity and increased risk of cardiovascular morbidity and mortality . Preventable or treatable risk factors in the initiation and development of pad include cigarette smoking, type 2 diabetes, hypertension, and hypercholesterolemia, which together may account for 75% of the excess pad risk . Besides these traditional risk factors, kidney function has also been associated with incident pad risk in a number of studies and is recognized as a pad risk factor . The clinical standard for the assessment of kidney function is the creatininebased estimated glomerular filtration rate (egfr). However, creatinine (molecular weight of 113 da) is a byproduct of muscle breakdown and tends to overestimate the egfr in individuals with lower creatinine generation due to loss of muscle mass (such as those with pad). In addition, renal tubules actively secrete creatinine, further reducing the value of creatinine to estimate gfr . Both 2microglobulin, a subunit of the major histocompatibility class i complex, and cystatin c, a cysteine protease inhibitor, have been proposed as alternative markers for the detection of renal impairment . Owing to their relatively low molecular weight compared with, for example, albumin (66 kda), 2microglobulin (12 kda) and cystatin c (13 kda) pass freely through the glomerular filtration barrier . Furthermore, these filtration markers are produced at a relatively constant rate and are neither affected by muscle mass nor subject to tubular secretion . In previous observational studies, both cystatin c and 2microglobulin have been shown to predict cardiovascular events and mortality more strongly than creatinine . However, evidence for an association between cystatin c and future risk of pad is sparse and conflicting . Also, although circulating 2microglobulin has previously been identified as a factor strongly linked to the presence and severity of pad in crosssectional studies, there are no studies that have prospectively examined this association . We, therefore, investigated the associations between 3 markers of kidney function (ie, 2microglobulin, cystatin c, and creatinine) with future risk of the development of pad in 2 large and wellcharacterized cohorts of men and women . The nurses health study (nhs) is a prospective cohort study of 121 701 female nurses aged 30 to 55 years at recruitment in 1976 . The health professionals followup study (hpfs) is a prospective cohort study of 51 529 male dentists, optometrists, pharmacists, podiatrists, osteopathic physicians, and veterinarians aged 40 to 75 years at recruitment in 1986 . Of these individuals, 32 826 women provided a blood sample in 1990 and 18 224 men provided blood specimens in 1994 . We excluded individuals who had a history of cardiovascular disease, including myocardial infarction; surgical / percutaneous revascularization of the coronary, carotid, or peripheral beds; confirmed pad; stroke; and transient ischemic attack . The casecontrol analytic datasets include all incident symptomatic pad cases, for a total of 144 cases and 432 controls in the nhs and 143 cases and 429 controls in the hpfs . Controls were matched 3:1 to cases on age, race, smoking history, month of blood draw (within 3 months), and fasting status, following the nested casecontrol design described by prentice and breslow . We selected controls at random, conditional on the matching factors, from participants free of cardiovascular diseases at the time the incident case occurred (risk set sampling). We restricted all cases and controls to those free of cardiovascular diseases at baseline to ensure identification of only incident cases . This included angina, myocardial infarction, surgical / percutaneous revascularization of the coronary, carotid, or peripheral beds, claudication, stroke, and transient ischemic attack, which are all assessed biennially . Because older age categories had fewer participants, we relaxed the age match range yearbyyear if necessary to a maximum of within 3 years . The institutional review boards of the brigham and women's hospital, harvard school of public health, and beth israel deaconess medical center approved the study protocol blood samples were shipped overnight with a cold pack to the central laboratory, centrifuged on arrival, aliquotted, and stored in liquid nitrogen at 130c to 196c . Nhs specimens were anticoagulated with heparin, and hpfs were anticoagulated with edta . Of all samples, 97% of those from the nhs and 95% of those from the hpfs were received within 24 hours; cystatin c and creatinine levels are known to remain stable in whole blood even when stored at room temperature up to 48 hours before separation . All analyses were performed in a laboratory certified by the centers for disease control and prevention / national heart, lung, and blood institute lipid standardization program with commercially available analytic systems . Measurements of 2microglobulin, cystatin c, and creatinine were performed on the roche p modular system (roche diagnostics) via an immunoturbidimetric technique (2microglobulin and cystatin c) or enzymatic method (creatinine), using reagents and calibrators from roche (2microglobulin and creatinine) or from genzyme (cystatin c). Interassay coefficients of variation for 2microglobulin, cystatin c, and creatinine were 5.9%, 3.0%, and 2.2% for nhs and 5.8%, 8.1%, and 6.5% for hpfs, respectively . The determination of highdensity lipoprotein cholesterol, lowdensity lipoprotein cholesterol, highsensitivity creactive protein (crp), triglycerides, and hemoglobin a1c has been described in greater detail elsewhere . Participants reported the occurrence of diagnosed medical conditions, including claudication and revascularization for arterial disease of the leg, during the previous 2 years on biennial mailed questionnaires . We collected medical records from treating physicians and hospitals for those who reported either condition . We defined pad as arterial disease below the aortic bifurcation (ie, excluding abdominal aortic aneurysm and renal artery stenosis). Confirmed pad required 1 of the following (in order of severity / certainty) in the medical record: (1) report of amputation, bypass, or other revascularization procedure for occlusive artery disease, (2) angiogram showing 50% stenosis of 1 artery with congruent symptoms in the ipsilateral limb, (3) anklebrachial index <0.90 at rest, or (4) physician's diagnosis . Questionnaires provided information about anthropometric data and lifestyle, including weight, detailed information about smoking, parental history of myocardial infarction, alcohol consumption, physical activity per week, medical history concerning medication use, and selfreported history of diabetes, hypertension, and hypercholesterolemia . Participants reported past smoking, time since quitting, and the average number of cigarettes smoked per day . Packyears were calculated as years of smoking multiplied by the average number of packs smoked per day and updated biennially . We calculated body mass index (bmi) by dividing weight (kg) by squared height (m). Physical activity was expressed as metabolic equivalent taskhours based on selfreported types and durations of activities over the previous year . Previous studies demonstrated that the validity and reproducibility of all these selfreported measures are high . We calculated the egfr based both on the creatininebased chronic kidney disease epidemiology collaboration (egfrckdepi) equation and on a more recent, combined creatinine continuous data are summarized as either meansd for normally distributed variables or median and interquartile range for nonnormally distributed variables . We used generalized linear mixed models and cochran mantel haenszel tests to compare continuous variables and categorical variables by casecontrol status, respectively, accounting for clustering by matching status . Associations between 2microglobulin, cystatin c, and creatinine and pad risk factors were examined using ageadjusted spearman partial correlation coefficients . To examine the prospective association of 2microglobulin, cystatin c, and creatinine with subsequent pad risk, we treated these markers as both a categorical (in quartiles) and a continuous (per 1sd increase in logtransformed marker) variable . Logtransformation of the markers led to a superior model fit . To test for a linear trend in the categorical analysis we also examined the possibility of nonlinear associations between the 3 biomarkers and pad nonparametrically with restricted cubic splines . In tests for nonlinearity, we used the likelihood ratio test, comparing the model with only the linear term to the model with the linear and the cubic spline terms . We used conditional logistic regression to estimate the odds ratio (or) and 95% ci for pad associated with increasing concentrations of these markers . Because the controls were selected using risk set sampling, the ors derived from conditional logistic regression provide unbiased estimates of the incidence rate ratio as a measure of relative risk (rr). We included covariables in our models as linear variables if appropriate or as categorical if they were discrete or their association with pad was nonlinear . Analyses that defined hypertension based on either a physician's diagnosis or a patient's antihypertensive medication use (thiazides, blockers, calcium channel blockers, and angiotensinconverting enzyme or angiotensin ii receptor antagonists) yielded similar results . To pool the rr estimates for women and men, we used metaanalyses with fixed effects or random effects when the test for heterogeneity was significant . We tested interactions with crossproduct terms between (continuous) logtransformed concentrations of the kidney function markers and selected variables in multivariableadjusted conditional or unconditional logistic regression models with additional adjustment for the matching factors . All analyses were performed using sas statistical software (version 9.3; sas institute). The nurses health study (nhs) is a prospective cohort study of 121 701 female nurses aged 30 to 55 years at recruitment in 1976 . The health professionals followup study (hpfs) is a prospective cohort study of 51 529 male dentists, optometrists, pharmacists, podiatrists, osteopathic physicians, and veterinarians aged 40 to 75 years at recruitment in 1986 . Of these individuals, 32 826 women provided a blood sample in 1990 and 18 224 men provided blood specimens in 1994 . We excluded individuals who had a history of cardiovascular disease, including myocardial infarction; surgical / percutaneous revascularization of the coronary, carotid, or peripheral beds; confirmed pad; stroke; and transient ischemic attack . The casecontrol analytic datasets include all incident symptomatic pad cases, for a total of 144 cases and 432 controls in the nhs and 143 cases and 429 controls in the hpfs . Controls were matched 3:1 to cases on age, race, smoking history, month of blood draw (within 3 months), and fasting status, following the nested casecontrol design described by prentice and breslow . We selected controls at random, conditional on the matching factors, from participants free of cardiovascular diseases at the time the incident case occurred (risk set sampling). We restricted all cases and controls to those free of cardiovascular diseases at baseline to ensure identification of only incident cases . This included angina, myocardial infarction, surgical / percutaneous revascularization of the coronary, carotid, or peripheral beds, claudication, stroke, and transient ischemic attack, which are all assessed biennially . Because older age categories had fewer participants, we relaxed the age match range yearbyyear if necessary to a maximum of within 3 years . The institutional review boards of the brigham and women's hospital, harvard school of public health, and beth israel deaconess medical center approved the study protocol blood samples were shipped overnight with a cold pack to the central laboratory, centrifuged on arrival, aliquotted, and stored in liquid nitrogen at 130c to 196c . Nhs specimens were anticoagulated with heparin, and hpfs were anticoagulated with edta . Of all samples, 97% of those from the nhs and 95% of those from the hpfs were received within 24 hours; cystatin c and creatinine levels are known to remain stable in whole blood even when stored at room temperature up to 48 hours before separation . All analyses were performed in a laboratory certified by the centers for disease control and prevention / national heart, lung, and blood institute lipid standardization program with commercially available analytic systems . Measurements of 2microglobulin, cystatin c, and creatinine were performed on the roche p modular system (roche diagnostics) via an immunoturbidimetric technique (2microglobulin and cystatin c) or enzymatic method (creatinine), using reagents and calibrators from roche (2microglobulin and creatinine) or from genzyme (cystatin c). Interassay coefficients of variation for 2microglobulin, cystatin c, and creatinine were 5.9%, 3.0%, and 2.2% for nhs and 5.8%, 8.1%, and 6.5% for hpfs, respectively . The determination of highdensity lipoprotein cholesterol, lowdensity lipoprotein cholesterol, highsensitivity creactive protein (crp), triglycerides, and hemoglobin a1c has been described in greater detail elsewhere . Participants reported the occurrence of diagnosed medical conditions, including claudication and revascularization for arterial disease of the leg, during the previous 2 years on biennial mailed questionnaires . We collected medical records from treating physicians and hospitals for those who reported either condition . We defined pad as arterial disease below the aortic bifurcation (ie, excluding abdominal aortic aneurysm and renal artery stenosis). Confirmed pad required 1 of the following (in order of severity / certainty) in the medical record: (1) report of amputation, bypass, or other revascularization procedure for occlusive artery disease, (2) angiogram showing 50% stenosis of 1 artery with congruent symptoms in the ipsilateral limb, (3) anklebrachial index <0.90 at rest, or (4) physician's diagnosis . Questionnaires provided information about anthropometric data and lifestyle, including weight, detailed information about smoking, parental history of myocardial infarction, alcohol consumption, physical activity per week, medical history concerning medication use, and selfreported history of diabetes, hypertension, and hypercholesterolemia . Participants reported past smoking, time since quitting, and the average number of cigarettes smoked per day . Packyears were calculated as years of smoking multiplied by the average number of packs smoked per day and updated biennially . We calculated body mass index (bmi) by dividing weight (kg) by squared height (m). Physical activity was expressed as metabolic equivalent taskhours based on selfreported types and durations of activities over the previous year . Previous studies demonstrated that the validity and reproducibility of all these selfreported measures are high . We calculated the egfr based both on the creatininebased chronic kidney disease epidemiology collaboration (egfrckdepi) equation and on a more recent, combined creatinine continuous data are summarized as either meansd for normally distributed variables or median and interquartile range for nonnormally distributed variables . We used generalized linear mixed models and cochran mantel haenszel tests to compare continuous variables and categorical variables by casecontrol status, respectively, accounting for clustering by matching status . Associations between 2microglobulin, cystatin c, and creatinine and pad risk factors were examined using ageadjusted spearman partial correlation coefficients . To examine the prospective association of 2microglobulin, cystatin c, and creatinine with subsequent pad risk, we treated these markers as both a categorical (in quartiles) and a continuous (per 1sd increase in logtransformed marker) variable . Logtransformation of the markers led to a superior model fit . To test for a linear trend in the categorical analysis we also examined the possibility of nonlinear associations between the 3 biomarkers and pad nonparametrically with restricted cubic splines . In tests for nonlinearity, we used the likelihood ratio test, comparing the model with only the linear term to the model with the linear and the cubic spline terms . We used conditional logistic regression to estimate the odds ratio (or) and 95% ci for pad associated with increasing concentrations of these markers . Because the controls were selected using risk set sampling, the ors derived from conditional logistic regression provide unbiased estimates of the incidence rate ratio as a measure of relative risk (rr). We included covariables in our models as linear variables if appropriate or as categorical if they were discrete or their association with pad was nonlinear . Analyses that defined hypertension based on either a physician's diagnosis or a patient's antihypertensive medication use (thiazides, blockers, calcium channel blockers, and angiotensinconverting enzyme or angiotensin ii receptor antagonists) yielded similar results . To pool the rr estimates for women and men, we used metaanalyses with fixed effects or random effects when the test for heterogeneity was significant . We tested interactions with crossproduct terms between (continuous) logtransformed concentrations of the kidney function markers and selected variables in multivariableadjusted conditional or unconditional logistic regression models with additional adjustment for the matching factors . All analyses were performed using sas statistical software (version 9.3; sas institute). Baseline characteristics of pad cases and controls are shown in table 1 . As expected, both female and male cases had higher levels of crp, triglycerides, and hemoglobin a1c and were more likely to have a history of diabetes, hypertension, or hypercholesterolemia than were matched controls . In men, 2microglobulin and cystatin c levels were significantly higher among cases than among controls, but in women, 2microglobulin and cystatin c levels did not differ between cases and controls . Creatinine was higher in male cases but lower in female cases compared to their matched controls . Based on egfr categories of the kidney disease: improving global outcomes (kdigo) clinical practice guideline, 66% of the women and 50% of the men had a normal or high kidney function (egfrckdepi 90 ml min per 1.73 m) and 33% and 46%, respectively, had a mildly reduced kidney function (egfrckdepi between 60 and 89 ml min per 1.73 m). Baseline characteristics of women and men who developed symptomatic peripheral artery disease during followup (cases) and matched controls matching factors, besides age, race, and smoking status, included date of blood draw and fasting status . Data are expressed as mean (sd), median (interquartile range), or number (percentage). P values are derived from mixed linear models for continuous variables and cochran mantel haenszel tests for categorical variables controlling for matched sets . Egfr indicates estimated glomerular filtration rate; hdl, highdensity lipoprotein; ldl, lowdensity lipoprotein; meth, metabolic equivalent hours . Cystatin c and 2microglobulin were strongly correlated with each other (=0.74, p<0.001 for women; =0.77, p<0.001 for men) (table 2) and highly correlated with creatinine, although not as strongly as with each other . Both 2microglobulin and cystatin c were correlated with age and modestly with bmi, crp, and triglycerides but not with packyears of smoking in both men and women . Besides an inverse correlation with packyears of smoking, no consistent correlations were observed between creatinine and cardiovascular risk factors in men and women . Both unadjusted spearman's rank correlation coefficients and pearson productmoment correlation coefficients are shown in table 3 . Spearman partial correlation coefficients between 2microglobulin, cystatin c, and creatinine and selected cardiovascular risk factors among 431 control women and 429 control men correlations are ageadjusted except age . Hdl indicates highdensity lipoprotein; ldl, lowdensity lipoprotein . * p<0.05, p<0.01, p<0.001 . Spearman's rank correlation coefficients and pearson productmoment correlation coefficients between 2microglobulin, cystatin c, and creatinine and selected cardiovascular risk factors among 431 control women and 429 control men hdl indicates highdensity lipoprotein; ldl, lowdensity lipoprotein . * p<0.05, p<0.01, p<0.001 . During a median followup of 15 years (interquartile range 11 to 17 years), pad was detected in 144 women, and during a median followup of 7 years (interquartile range 4 to 10 years) pad was detected in 143 men . Of the 144 pad cases among women, 74 (51%) cases were confirmed by medical records indicating surgery or revascularization procedures, 24 (17%) cases by angiogram, 39 (27%) cases by anklebrachial index, and 7 (5%) cases by physician diagnosis . Of the 143 pad cases among men, medical reports of amputation, bypass, or other revascularization procedure confirmed 87 (61%) pad cases; angiogram or doppler ultrasound, 15 (10%) cases; anklebrachial index <0.90, (16%) 23 cases; and physician diagnosis, 18 (13%) cases . Because there was no evidence for nonlinear associations between markers of kidney function and future risk of pad, we conducted analyses per 1sd increase in logtransformed markers . Higher circulating levels of 2microglobulin, cystatin c, and creatinine were associated with increased risk of symptomatic pad in men but not in women (table 4). Rrs among women tended to be higher for 2microglobulin than the other 2 measures, and the difference between men and women was significant for cystatin c but not for 2microglobulin (figure). Although expected given the limited variation in age, sex, and ethnicity (the main determinants of egfrckdepi besides creatinine) between cases and matched controls, the associations with risk of pad were similar if kidney function was estimated using the egfrckdepi formula rather than using circulating creatinine (table 5). Associations were also similar when we modeled the inverse of 2microglobulin or cystatin c (table 5), as surrogate of egfr, compared with the logtransformed values of 2microglobulin or cystatin c, respectively . Relative risks for peripheral artery disease according to 2microglobulin, cystatin c, and creatinine in women and men hdl indicates highdensity lipoprotein; ldl, lowdensity lipoprotein . Matching factors included age, race, smoking status, month of blood draw, and fasting status . Adjusted for creactive protein, hdl cholesterol, ldl cholesterol, hemoglobin a1c, triglycerides, packyears of smoking, type 2 diabetes, hypertension, hypercholesterolemia, parental history of myocardial infarction before age 60 years, body mass index, physical activity, and alcohol consumption . Relative risks for peripheral artery disease according to egfrckdepi, egfrcreatcys, inverse of 2microglobulin, and inverse of cystatin c in women and men ckdepi indicates chronic kidney disease epidemiology collaboration; creatcys, creatinine cystatin c; egfr, estimated glomerular filtration rate; hdl, highdensity lipoprotein; ldl, lowdensity lipoprotein . P for trend based on median of quartiles . Matching factors included age, race, smoking status, month of blood draw, and fasting status . Adjusted for hdl cholesterol, ldl cholesterol, creactive protein, hemoglobin a1c, triglycerides, packyears of smoking, type 2 diabetes, hypertension, hypercholesterolemia, parental history of myocardial infarction before age 60 years, body mass index, physical activity, and alcohol consumption . Adjusted relative risks (95% ci) for peripheral artery disease per 1sd increment in 2microglobulin, cystatin c, and creatinine . Relative risks are adjusted for creactive protein; hdl and ldl cholesterol; hemoglobin a1c; triglycerides; creatinine (except creatinine, which is adjusted for 2microglobulin instead); packyears of smoking; history of type 2 diabetes, hypertension, and hypercholesterolemia; parental history of myocardial infarction before age 60 years; body mass index; physical activity; and alcohol consumption . Matching factors are age, race, smoking status, month of blood draw, and fasting status . To calculate the pooled estimates, we used metaanalyses with fixed effects or random effects in case of significant heterogeneity (p<0.05). Hdl indicates highdensity lipoprotein; hpfs, health professionals followup study; ldl, lowdensity lipoprotein; nhs, nurses health study . Although the rrs increased slightly after further accounting for circulating creatinine, 2microglobulin and cystatin c remained unassociated with pad risk among women (figure). For comparison, the corresponding multivariableadjusted rr for each 1sd increment in crp was 1.48 (95% ci 1.14 to 1.94) in women and 1.39 (95% ci 1.08 to 1.79) in men . In exploratory analyses when models were mutually adjusted for both 2microglobulin and cystatin c, the rrs per each 1sd increment were 1.44 (95% ci 0.94 to 2.22) for 2microglobulin and 0.73 (0.48 to 1.12) for cystatin c in women and 1.31 (95% ci, 0.84 to 2.03) for 2microglobulin and 1.26 (0.77 to 2.06) for cystatin c in men . In further analyses, we found no interactions of 2microglobulin or cystatin c with creatinine, conventional cardiovascular risk factors, or duration of followup in women and men (table 6). P for interaction values between 2microglobulin and cystatin c and selected risk factors in the association with symptomatic pad hdl indicates highdensity lipoprotein; ldl, lowdensity lipoprotein . In pooled analyses of men and women, the rrs per each 1sd increment were 1.31 (95% ci 1.04 to 1.64) for 2microglobulin and 1.19 (95% ci 0.74 to 1.93) for cystatin c in multivariableadjusted models including adjustment for creatinine (figure). When we adjusted for egfrckdepi instead of circulating creatinine, the pooled rrs for 2microglobulin (rr 1.28, 95% ci 1.02 to 1.60) and cystatin c (rr 1.13, 95% ci 0.89 to 1.43) remained essentially the same . Similar results were obtained for 2microglobulin (rr 1.33, 95% ci 1.05 to 1.70) and for cystatin c (rr 1.20, 95% ci 0.73 to 1.96) when we excluded subjects with an egfrckdepi <60 ml min per 1.73 m. the pooled multivariableadjusted estimate for creatinine (rr 1.09, 95% ci 0.73 to 1.64) decreased further after additionally accounting for either 2microglobulin (rr 0.94, 95% ci 0.77 to 1.15) or cystatin c (rr 0.98, 95% ci 0.79 to 1.21). In 2 large nested casecontrol studies, circulating 2microglobulin and cystatin c and, to a lesser extent, creatinine were associated with incident pad in men, although creatinine was no longer associated with risk after adjustment for either 2microglobulin or cystatin c. circulating cystatin c and creatinine were completely unassociated with risk of pad among women . In pooled analyses among men and women, the association of 2microglobulin with pad risk remained after accounting for circulating creatinine and for other established cardiovascular risk factors . Moreover, 2microglobulin was also associated with pad risk in individuals with egfrs above the clinical cutoff for chronic kidney disease (egfrckdepi 60 ml min per 1.73 m), extending the possible range over which kidney function is related to risk . Using highthroughput proteomic profiling, a crosssectional study that included both men and women demonstrated that 2microglobulin correlated strongly with pad severity, independent of other risk factors . Our present study complements and extends this finding by showing a prospective association between 2microglobulin and future risk of symptomatic pad among subjects free of diagnosed cardiovascular or kidney disease . This increased risk of systemic atherosclerosis concords with positive associations between 2microglobulin and risk of coronary heart disease in postmenopausal women and in the general population . Among japanese our data indicate that the association of 2microglobulin with incident symptomatic pad is largely independent of circulating creatinine or creatininebased egfr . This may suggest that 2microglobulin represents kidney function that is not well captured by circulating creatinine . Indeed, 2microglobulin is correlated more strongly with gfr measured by inulin than is serum creatinine . Another possibility that cannot be excluded by the present data is that 2microglobulin reflects other, nongfr determinants of pad risk . Given the modest correlations with crp, 2microglobulin may be a marker of local or systemic inflammation, but the observed association with pad remained after adjustment for crp . In addition, it is possible that circulating 2microglobulin reflects amyloid deposition and aggregation in the vessel wall as part of the atherosclerotic process . Perhaps most intriguingly, the repetitive ischemia reperfusion injury of the limbs that occurs as pad develops may promote 2microglobulin shedding from endothelial cell surfaces due to its noncovalent binding with the major histocompatibility complex class i chain without direct attachment to the cell membrane . Few prospective studies have investigated the association of cystatin c with risk of incident pad among apparently healthy subjects . In> 4000 communitydwelling elderly subjects of the cardiovascular health study, cystatin c was associated with an increased risk of lower extremity pad procedures (bypass surgery, angioplasty, or amputation) only among subjects in the highest quintile (threshold for cystatin c> 1.27 mg / l). In a nested casecontrol study with 133 symptomatic pad cases in the physicians health study, no evidence was found for an increased risk with elevated cystatin c levels . However, the 95th cutoff percentile for cystatin c based on the distribution of the controls was <1.08 mg / l . Although we did not find evidence for a nonlinear association, the higher pad risk in our study among men appeared to be statistically significant when comparing the first (<0.83 mg / l) cystatin c quartile with the fourth (> 1.10 mg / l), but no such association was observed in women . These results suggest that only particularly high levels of cystatin c may be associated with increased risk of pad, although differences between studies hamper direct comparisons . First, determination of 2microglobulin is already widely available and routinely measured in clinical settings for hematological and lymphoproliferative diseases (eg, multiple myeloma and lymphoma). Measurement of 2microglobulin for the early detection of pad may thus be easily incorporated into routine care cardiovascular risk prediction . Of note, measurement of cystatin c second, these findings suggest that (preservation of) kidney function may be an additional target to reduce the risk of pad . It cannot be excluded that lower kidney function may be a consequence rather than a cause of atherosclerosis, particularly if renal arteries occlude in parallel with arteries of the lower extremities . The observation, however, that kidney function is associated with pad risk even after extensive adjustment for cardiovascular risk factors suggests a role for kidney function itself as pad risk promoter . Relative risks of all 3 markers of kidney function tended to be higher in the hpfs (men) than in the nhs (women). Besides sex first, plasma was stored in heparin tubes for nhs samples versus in edta tubes for hpfs samples, which may have created differential measurement of these markers . This is unlikely, though, because baseline correlations between these markers with each other and with other confounding variables were almost exactly the same for the women in nhs as for the men in hpfs . Also, the coefficients of variation of the measurements for all 3 markers were relatively low for both hpfs and nhs . It is also unlikely that differences in end point definitions may explain the stronger associations because definitions were similar between the 2 studies and positive associations of comparable magnitude between wellknown pad risk factors, such as for crp and lipoprotein(a), were observed in both studies . Third, egfr was systematically higher in women, and in these analyses, only 2microglobulin tended to be positively associated with risk . This suggests that it may provide more useful discrimination at high levels of egfr than other markers of kidney function . Nevertheless, the stronger associations among men could reflect differences in pad pathophysiology between sexes, although a recent metaanalysis indicated that there is no reason to assume that kidney function is a less important risk factor for other forms of cardiovascular disease in women than in men . Future studies should address whether markers of kidney function universally perform better in men or whether 2microglobulin is a superior marker to assess risk of the development of pad . Strengths of the current study are the large number of biochemical and traditional cardiovascular risk factors for which we could adjust in our analyses, the prospective design, the longterm followup, the use of risk set sampling to select controls, the homogeneity of the 2 study populations, and the use of adjudicated events with a pronounced atherosclerotic origin . Besides these strengths, several potential limitations warrant to be mentioned . Subclinical or asymptomatic pad, which may have otherwise been detected through an abnormal anklebrachial index or pulse examination, is likely to have been missed . However, outcomes included in this analysis were confirmed by medical records, reducing the likelihood of falsepositive cases at the expense of a risk of a falsely low incidence rate . Second, participants in both cohorts were predominantly white; extrapolation of our results to other race / ethnicity subgroups should therefore be done with caution . Blacks, for instance, have higher circulating creatinine but not 2microglobulin or cystatin c levels and are at higher risk of the development of pad . Therefore, it is impossible to know if the differences in significance between men and women were due to differences in study design or true sex differences . Fourth, although 2microglobulin and cystatin c clearly outperformed creatinine in assessing pad risk, particularly among the men, we cannot directly determine whether that reflects better estimation of gfr or simply nonrenal effects . Fifth, although crp was strongly associated with risk in both cohorts, we adjusted for only crp, which may be an imperfect measure of inflammation . Finally, we did not have data on albuminuria, which could potentially have captured other aspects of chronic kidney disease . The present study identified an association between circulating 2microglobulin and future risk of symptomatic pad in pooled analyses of men and women . When the 2 sexes were studied separately, 2microglobulin and cystatin c were associated with risk of symptomatic pad in men but not in women . The associations of these emerging markers of kidney function with pad risk appeared to be independent of circulating creatinine or creatininebased egfr, which is the current clinical standard for the assessment of kidney function and wellknown cardiovascular risk factors . Cystatin c and particularly 2microglobulin may have greater potential than creatinine for the early detection of systemic atherosclerotic diseases . Appendix beta2-microglobulin, cystatin c, and creatinine and risk of symptomatic peripheral artery disease.
Multiple myeloma is a malignant neoplasm that is characterized by a monoclonal proliferation of plasma cells . The clinical manifestations of the disease occur as a result of an expanding plasma cell mass in the bone marrow and other factors produced by these cells such as monoclonal immunoglobulin, bence - jones proteins and osteoclast activating factors . The common clinical signs and symptoms of multiple myeloma include pain in the bone, fatigue, anemia and infectious diseases . Oral and maxillofacial manifestations as an initial sign or symptom of multiple myeloma are rare . In 12 - 15% of cases, oral involvement can be apparent as swelling, orofacial pain, mobility of teeth, numbness and paresthesia, hemorrhage, fracture and root resorption . We hereby present a case of multiple myeloma with first clinical manifestation as generalized gingival enlargement . A 58-year - old male patient was referred to our department because of the generalized enlargement of gingiva . The enlargement was first noted by the patient 6 months prior to the referral, and progressed steadily since then . The intraoral examination revealed soft, granular, friable, non - tender, and red / magenta enlargement that bled spontaneously . The enlargements were present on both buccal and lingual / palatal sides [figure 1]. On physical examination, grade iii mobility was observed in 16, 17, 18, 36, 37 . The medical history of the patient included epilepsy for which he took prescribed medication with no occurrence of seizure for last 10 years . Based on clinical presentation, a provisional diagnosis of inflammatory gingival enlargement routine blood investigations were done along with hiv and hepatitis b and sputum examination to rule out any leukemic infiltration and enlargement associated with tuberculosis . Orthopantomography was advised, which revealed severe bone loss in 16, 17, 18 and 36, 37 regions . After phase i therapy and consultation with the physician, gingivectomy was performed in anterior mandibular region and excised tissue was sent for histopathological examination . The patient was followed up every week [figure 2] but after 1 month the clinical examination revealed recurrence of enlargement in the anterior mandibular region [figure 3]. The subepithelial zone showed infiltration by sheets of plasma cells mainly mature with few being less differentiated [figure 4]. Based on these findings, serum protein electrophoresis and urine analysis for bence - jones proteins was carried out . Subsequently, patient was advised to undergo cranial and pelvic radiography [figures 6 and 7]. The patient was diagnosed as a case of multiple myeloma and chemotherapy was started with thalidomide . Gingiva 10 days after excisional biopsy was taken recurrence after 1 month of biopsy areas of ulceration and sheets of plasma cells multinucleate and binucleate plasma cells pelvic radiograph showing osteolytic lesions skull radiograph showing osteolytic lesions normal gingiva 1 month after chemotherapy multiple myeloma is the most aggressive plasma cell neoplasia and most common primary malignancy of bone . It has a predilection for areas of active hematopoiesis such as the lumbar spine, ribs, and pelvic bones . Jawbone involvement in multiple myeloma is common and often occurs in the advanced stages of the disease . Jaw involvement in multiple myeloma was reported by bruce and royer to have a prevalence rate of 28.8% (17 of 59 total cases). Epstein et al ., reported that 14.1% of 783 multiple myeloma cases had oral manifestations . Lesions such as swelling, orofacial pain, mobility of teeth, numbness and paresthesia, hemorrhage, fracture and root resorption are more frequently found in the mandible than in the maxilla, especially in the posterior third and angle of the jaw, perhaps because of greater hematopoietic activity in these areas . A radiographic survey of patients with multiple myeloma reveals multiple well - defined punched out radiolucencies involving bone . These radiolucent areas of bone contain the abnormal plasma cell proliferations that characterize the disease . In our case, type 2 bone involvement was seen, which is by far, the most common presentation of multiple myeloma . In conclusion, dental surgeons can play an important role in the early recognition of oral lesions with underlying systemic disease, thus preventing the morbidity and mortality associated with such pathologies.
Gliomas are the most common primary brain tumors in adults and are usually classified and graded according to the world health organisation (who). Currently, chemotherapy is standard of care for all diffuse gliomas, either at first diagnosis or at first recurrence [15]. Treatment options for patients failing radiotherapy and a first line of alkylating or methylating chemotherapy are limited . The cytotoxic effect of tmz is mediated primarily via methylation at the o position of guanine . One of the main mechanisms of tumor resistance to tmz is thought to be mediated by o - methylguanine - dna methyltransferase (mgmt). Evidence supporting this role of mgmt comes from clinical studies indicating that hypermethylation of the promoter of mgmt is associated with improved tumor response and survival in patients with gbm [7, 8]. Because of the more continuous exposition with ddtmz it has been assumed that dose dense temozolomide (ddtmz) schedules could overcome mgmt dependent resistance against tmz by a more effective depletion of deplete intracellular levels of the dna repair enzyme, mgmt . Studies using ddtmz show it is well tolerated and generally safe, also when given in higher monthly doses and in patients who have previously received tmz [1018]. We used the 1 week on/1 week off tmz regimen (ddtmz) for patients with relapsing gbm or other glioma after prior tmz or nitrosourea chemotherapy to study the efficacy and toxicity of ddtmz in heavily pre - treated patients with high - grade glioma . Data of all diffuse glioma patients treated with ddtmz after prior chemotherapy in our center were retrospectively collected . Patients were included in this study if they had a histologically confirmed low - grade glioma or high - grade glioma, with a progressive and measurable enhancing tumor on the mri (diameter> 2 cm), relapsing after prior radiotherapy and at least one line of chemotherapy, and had concluded rt at least 3 months prior to the diagnosis of progression . We collected data about patient characteristics, tumor characteristics, prior treatment, number of ddtmz cycles, use of dexamethason, adverse effects, reason of discontinuation, and further treatments . According to histology three categories of patients were distinguished: patients treated with ddtmz for a progressive primary gbm after radiotherapy and tmz (group a); patients with recurrent astrocytoma who grade 2 or 3 (group b), or recurrent oligo - astrocytoma who grade 2 or 3, without 1p and 19q loss; and patients with progressive who grade 2 or 3 oligodendroglioma or oligo - astrocytoma with 1p and 19q loss (group c). Who grade 2 tumors were combined with who grade 3 tumors because all patients had contrast enhancing lesions on the mri scan at the time of treatment with ddtmz, suggesting malignant dedifferentiation of the who grade 2 tumors . Furthermore, a previous study with tmz in recurrent who grade 2 astrocytoma, with enhancement on the mri - scan, at our institution has shown similar results to the pilot trial of tmz in recurrent who grade 3 gliomas (pfs at 12 months 25% vs. 24%). [3, 4]. Patients received tmz on day 17 and on day 1521 of a 28-day cycle for up to 12 cycles or until documented disease progression or unacceptable toxicity . The starting dose of the tmz was 100 mg / m / day . In the absence of toxicity or only ctcae grade 1 toxicity during the first two treatment weeks the dose was escalated, in two steps to dose level 1 (150 mg / m / day; table 1 for dose levels). Toxicity was evaluated according to the common terminology criteria for adverse events (ctcae; version 3.0). In case of hematological toxicity grade 4 or non - hematological toxicity grade 3, the dosage tmz of the next cycle was reduced with 1 dose level . In case of ctcae grade 4 non - hematological toxicity, 4 hematological toxicity or a grade 3 non - hematological toxicity at dose level 4 (75 mg / m / day), the patient went off treatment . In case of dose reductions, blood examinations were done on day 15 and day 29 and when platelets were above 100 * 10/l and neutrophils counts above 1.5 * 10/l, the following 7 days tmz was administered . Otherwise treatment was postponed until recovery to ctcae grade 1 and/or platelets were above 100 * 10/l . The treatment was stopped if it had to be postponed for more than 2 weeks.table 1dose levels of dose dense temozolomidedose leveldaily temozolomide dose (mg / m / day)dose temozolomide per cycle (mg / m)1150210021251750310014004751050 dose levels of dose dense temozolomide the objectives of the study were the assessment of progression free survival at 6 (pfs-6) and 12 (pfs-12) months, objective response rate (orr), overall survival (os), and toxicity . Os was calculated from the start of the tmz treatment to the date of death . Pfs was calculated from the start of the tmz until the date of progression or death . Clinical evaluation was done every 4 weeks and mri was made every 12 weeks or in case of neurological deterioration . Response to treatment was reviewed as part of this analysis (w.t . ). In this exploratory analysis, between june 2005 and june 2011, 53 patients were treated with ddtmz for the progression of a glioma in our center . Twenty - four patients were treated for a recurrent gbm (group a), 16 patients were treated for a recurrence of a who grade 2 or 3 astrocytoma, oligodendroglioma, or oligo - astrocytoma, without combined 1p and 19q loss, with a contrast enhancing lesion on mri (group b) and 13 patients were treated for a recurrence of a who grade 2 or 3 oligodendroglioma or oligo - astrocytoma with combined 1p and 19q loss, with a contrast enhancing lesion on mri (group c). All gbm patients progressed after chemo - irradiation with tmz, except for one patient who had progression after radiotherapy and during the 6th cycle of 1st line standard day 15 every 4 weeks schedule tmz chemotherapy . Five gbm patients treated with chemo - irradiation progressed directly after six cycles of adjuvant tmz; all other patients had a tmz free interval, before the start of ddtmz . Six patients with a recurrent primary - gbm received a second line of therapy after chemo - irradiation: dendritic - cell therapy (1), cediranib (1), lomustine combined with cediranib (2), and, imatinib combined with hydroxyurea (2).table 2characteristics of patients treated with dose dense temozolomide for a progressive glioma after radiotherapy and 1 or 2 lines of chemotherapycharacteristicall patientsno . (%) of patients, n = 53group ano . (%) of patients, n = 24group bno . (%) of patients, n = 16group cno . (%) of patients, n = 13age, years median49524344 range3174317432613360sex male38 (72%)14 (58%)13 (81%)11(85%) female15 (28%)10 (42%)3 (19%)2 (15%)first symptom epilepsy36 (68%)11 (46%)13 (81%)12 (92%) other17 (32%)13 (54%)3 (19%)1 (8%)who - ps 015 (28%)6 (25%)5 (31%)4 (31%) 127 (51%)16 (67%)7 (44%)4 (31%) 211 (21%)2 (8%)4 (25%)5 (38%)who histology grade at first operation 210 (63%)6 (46%) 36 (37%)7 (54%)who world health organisation, ps performance scoregroup a patients with recurrent primary glioblastoma, group b patients with recurrent who grade 2 or 3 astrocytoma or oligodendroglioma or oligoastrocytoma without combined 1p and 19q loss and with a contrast enhancing lesion on mri, group c patients with recurrent who grade 2 or 3 oligodendroglioma or oligoastrocytoma with combined 1p and 19q loss and with a contrast enhancing lesion on mritable 3previous treatments of patients treated with dose dense temozolomide for a progressive glioma after radiotherapy and 1 or 2 lines of chemotherapycharacteristicall patientsno . (%) of patients, n = 53group ano . (%) of patients, n = 24group bno . (%) of patients, n = 16group cno . (%) of patients, n = 13ddtmz as 2nd line of chemotherapy40 (75%)19 (79%)11 (69%)10 (77%)ddtmz as 3nd line of chemotherapy13 (25%)5 (21%)5 (31%)3 (23%)second operation15 (28%)3 (13%)6 (38%)6 (46%)third operation1 (2%)001 (8%)time between start last rt and start ddtmz, median (range) in months24 (5197)19 (875)18 (5197)45 (1093)time between last chemotherapy and start ddtmz, median (range) in months10 (094)5 (067)13 (192)17 (194)prior 1st line treatment53 (100%)24 (100%)16 (100%)13 (100%)rt / tmz27 (51%)23 (96%)3 (19%)1 (8%)tmz6 (11%)1 (4%)5 (31%)0pcv20 (38%)08 (50%)12 (92%)prior 2nd line treatment13 (25%)5 (21%)5 (31%)3 (23%)rt / tmz1 (2%)01 (6%)0tmz5 (9%)03 (19%)2 (15%)pcv2 (4%)01 (6%)1 (8%)other5 (9%)5 (21%)00ddtmz dose dense temozolomide, rt radiotherapygroup a patients with recurrent primary glioblastoma, group b patients with recurrent who grade 2 or 3 astrocytoma or oligodendroglioma or oligoastrocytoma without combined 1p and 19q loss and with a contrast enhancing lesion on mri, group c patients with recurrent who grade 2 or 3 oligodendroglioma or oligoastrocytoma with combined 1p and 19q loss and with a contrast enhancing lesion on mri characteristics of patients treated with dose dense temozolomide for a progressive glioma after radiotherapy and 1 or 2 lines of chemotherapy who world health organisation, ps performance score group a patients with recurrent primary glioblastoma, group b patients with recurrent who grade 2 or 3 astrocytoma or oligodendroglioma or oligoastrocytoma without combined 1p and 19q loss and with a contrast enhancing lesion on mri, group c patients with recurrent who grade 2 or 3 oligodendroglioma or oligoastrocytoma with combined 1p and 19q loss and with a contrast enhancing lesion on mri previous treatments of patients treated with dose dense temozolomide for a progressive glioma after radiotherapy and 1 or 2 lines of chemotherapy ddtmz dose dense temozolomide, rt radiotherapy group a patients with recurrent primary glioblastoma, group b patients with recurrent who grade 2 or 3 astrocytoma or oligodendroglioma or oligoastrocytoma without combined 1p and 19q loss and with a contrast enhancing lesion on mri, group c patients with recurrent who grade 2 or 3 oligodendroglioma or oligoastrocytoma with combined 1p and 19q loss and with a contrast enhancing lesion on mri the median number of cycles of ddtmz was 4 (range 112), three patients completed 12 cycles . Eight patients discontinued ddtmz because of toxicity: grade 4 thrombocytopenia (1), persistent grade 2 or grade 3 fatigue (5), grade 3 elevated transaminases (1), and grade 3 allergic skin reaction (1). Five patients who stopped because of fatigue continued tmz in regular regimen of day 15 in a 28 day cycle and tolerated this well . In 25 patients, cd4 + lymphocytes counts were monitored; 14 (56%) of these patients developed a grade 3 cd4 + lymphopenia (<0.2 * 10/l) and 6 (24%) of these patients developed a grade 4 cd4 + lymphopenia (<0.05 * 10/l). Two of the patients with a grade 4 cd4 + lymphopenia developed a pneumocystis carinii pneumonia, prior to routine monitoring of cd4 + counts, from which they fully recovered . The pfs-6, the orr (complete and partial response) and median os for the three groups of patients are shown in table 4 . The median interval (and range) between the prior chemotherapy and the start of the ddtmz was 5 months (067 months) in group a, 12.5 months (range 192) in group b and 17 months (range 194) in group c. the patients without 1p and 19q loss (group a and b) that started with the ddtmz within 3 months of the previous chemotherapy (12 out of 40 patients) had a lower pfs-6 compared to the patients with a chemotherapy free interval of more than 3 months (pfs-6 8% vs. 43%; fisher exact test 0.033).table 4outcome of patients treated with dose dense temozolomide for a progressive glioma after radiotherapy and 1 or 2 lines of chemotherapyoutcomeall patientsn = 53group an = 24group bn = 16group cn = 13pfs-640%29%38%62%pfs-1213%13%13%15%median os9 months6 months9 months19 monthscr + pr17%8%19%33% (4:12)pfs-6 progression free survival at 6 months, pfs-12 progression free survival at 12 months, os overall survival, cr complete response, pr partial responsegroup a patients with recurrent primary glioblastoma, group b patients with recurrent who grade 2 or 3 astrocytoma or oligodendroglioma or oligo - astrocytoma without combined 1p and 19q loss and with a contrast enhancing lesion on mri, group c patients with recurrent who grade 2 or 3 oligodendroglioma or oligo - astrocytoma with combined 1p and 19q loss and with a contrast enhancing lesion on mri outcome of patients treated with dose dense temozolomide for a progressive glioma after radiotherapy and 1 or 2 lines of chemotherapy pfs-6 progression free survival at 6 months, pfs-12 progression free survival at 12 months, os overall survival, cr complete response, pr partial response group a patients with recurrent primary glioblastoma, group b patients with recurrent who grade 2 or 3 astrocytoma or oligodendroglioma or oligo - astrocytoma without combined 1p and 19q loss and with a contrast enhancing lesion on mri, group c patients with recurrent who grade 2 or 3 oligodendroglioma or oligo - astrocytoma with combined 1p and 19q loss and with a contrast enhancing lesion on mri in this group of 53 chemotherapy and radiotherapy pre - treated gliomas, ddtmz showed activity . Although this is a retrospective study with a limited sample size, our results are also comparable to other studies on dose - dense tmz in recurrent gliomas [1217]. However, in all our groups the observed activity is well within the range of previous reports on standard dosing tmz . The pfs-6 of 29% (95%-ci 1147%) (table 4; group a) in gbm is within the range of the pivotal standard dose phase ii tmz trials in recurrent gbm . (pfs-6: 1924%) [2123] more in particular, brandes et al . Described a 24% (95% ci 1442%) for 2nd line standard dosing tmz in recurrent gbm . The results in group b (recurrent who grade 2 or 3 astrocytoma or oligodendroglioma without 1p and 19q loss) are comparable to the 2nd line results in the pivotal phase 2 trial in recurrent anaplastic astrocytoma or anaplastic oligo - astrocytoma (pfs-6 44% versus pfs-6 38% in the present series, table 4). The pfs-6 in group b and c (all recurrent non - primary gbm s, with a contrast enhancing lesion on mri at the start of the ddtmz) is higher than the pfs-6 found in the eortc study 26972 in recurrent oligodendroglioma, with or without combined 1p/19q loss after first line chemotherapy (table 4: pfs-6 3862% vs. 29%), although the pfs-12 is comparable (table 4: pfs-12 1315% vs. 11%). Data on second line tmz in recurrent oligodendroglial tumors with combined 1p/19q loss are scarce, kouwenhoven et al . Reported only one responder in nine patients treated after prior procarbazine, lomustine, and vincristine chemotherapy, but pfs-6 was not reported . Almost none of the patients with a primary gbm or who grade 2 or 3 glioma without combined 1p/19q loss (group a and b) with a chemotherapy free interval of 3 months or less before the start of the ddtmz had a good outcome (pfs-6 8% vs. 43%; fisher exact test 0.033). Similar to the results of perry et al . On metronomic tmz, ddtmz is not effective in patients with progressive disease within 3 months of previous chemotherapy . The patients with a dedifferentiated glioma with combined 1p/19q loss were left out of this analysis, because these patients have a completely different prognosis and response to chemotherapy and only two patients in this group had progressive disease within the 3 months before the start of the ddtmz . The single patient relapsing during standard tmz and responding to ddtmz had a progression free survival of 48 months up till now, suggesting he did nt have real tumor progression at the time of ddtmz . Probably the enhancement on mri in this patient was caused by radionecrosis 45 months after rt . Since all patients started ddtmz relatively long after (chemo-)irradiation (median time between start last rt and start ddtmz 24 months, range 5197; table 3), it is unlikely that pseudoprogression played a role in this study . Five patients were switched to the standard day 15 every 4 weeks tmz because of fatigue . After switch their fatigue improved . Two patients developed pcp infections before routine monitoring of cd4 + counts, none of the monitored patients (who received pcp prophylaxis with cotrimoxazol in case cd4 + counts decreased below 0.2 * 10/l) developed a pcp infection . Data from available phase 2 trials investigating ddtmz in gliomas indicate a high incidence of lymphopenia, especially in patient treated with the 3 weeks on/1 week off regimen [12, 14, 16, 17, 27]. In melanoma patients treated with daily tmz for 6 weeks out of every 8-week cycle, a high incidence of lymphopenia and an increased risk of opportunistic infections were reported . Clearly, patients who receive ddtmz are at risk to develop pneumocystis carinii pneumonia, and prophylaxis is indicated in patients who develop lymphopenia or low cd4 + counts . Although this study has a limited number of patients and is retrospective, it however seems from these results that ddtmz is an effective treatment for patients with a recurrence of gbm or otherwise heavily pre - treated gliomas, albeit with an increase in toxicity . Whether it is more effective than the standard 5 of 28-day regimen remains unclear . Although administration of ddtmz regimens causes more pronounced depletion of mgmt in peripheral blood mononuclear cells, the effects of ddtmz on mgmt activity in brain tumor tissue and its impact on clinical outcome remain unclear . A study from the united kingdom, comparing standard day 15 every 4 weeks tmz with a ddtmz schedule, (given in a 3 weeks on/1 week off schedule) failed to show any benefit of ddtmz in high - grade glioma recurrent after rt only in comparison to the standard day 15 every 4 weeks schedule . Of note, although these patients were chemotherapy nave, one may assume that two - thirds of patients would have an unmethylated mgmt promoter . Thus, if ddtmz would have been effective in overcoming that resistance, one would expect at least some trend toward a more favorable outcome in ddtmz treated patients . The recently reported rtog 0525 trial on newly diagnosed gbm also failed to produce superior outcome of ddtmz in newly diagnosed gbm (and regardless of the mgmt promoter status).
Chest pain is a typical symptom of acute coronary syndrome (acs), of which there are three common types: unstable angina (ua), st segment elevation (stemi) of myocardial infarction (mi), and non - st - segment elevation of mi (nstemi) [1, 2]. Symptoms of acs in the absence of chest pain are dyspnea, nausea, sweating, neck or jaw and arm pain . Ear pain and sore throat are rare symptoms of acs, which makes diagnosis and treatment difficult and challenging . We present a rare case of a patient presented to the emergency department of our hospital with symptoms of earache and sore throat for cardiac ischemia . A 53-year - old african - american female presented to the ed complaining of earache and sore throat for 3 days . She described the pain as a burning sensation that was worst when she woke up in the morning and got better as the day progressed . The patient denied chest pain, shortness of breath, nausea, vomiting, or dizziness . The patient worked as a telemarketer, described her job as very stressful, and claimed to smoke one to two cigarettes per week . Her medical history was significant for dyslipidemia, newly diagnosed type 2 diabetes, and chest discomfort diagnosed 4 months ago at a different hospital as stemi with complete occlusion of the rca . She was treated at the hospital by placement of a bare - metal stent in her rca . Current home medications included clopidogrel, aspirin, statin, beta - blocker, metformin, and nitroglycerin sublingually as needed for chest pain . Her vital signs on arrival at the ed were: bp of 131/82 mmhg, pulse of 70 beats per minute, respiratory rate of 17 breaths per minute, temperature of 98 f, and oxygen saturation of 98% on room air . Because of multiple risk factors, in addition to a symptom that could potentially be a referred cardiac pathology, an ecg, chest radiograph, and cardiac biomarkers were ordered at the triage . Cardiovascular, pulmonary, abdominal, ear, nose, and throat examinations were unremarkable . The initial ecg displayed no st segment elevation, but t wave inversion in inferior leads that was unchanged from a previous ecg . A presumptive diagnosis of acs/ nstemi was made on the basis of an elevated troponin combined with the ecg changes . After the acs protocol was initiated, a cardiology consultation was requested, and the patient was admitted to our hospital . On admission, the patient continued to complain of throbbing ear pain that fluctuated now between the two ears, but again denied chest pain or shortness of breath . On the 2nd day it was found that the previous bare - metal stent in the posterolateral branch (distal rca) was completely occluded, and no balloon could be advanced beyond that area . A new lesion in the proximal / ostial rca greater than 70% occlusion was opened with a successful placement of a pci at the ostial rca using a drug - eluting stent . Following this, her troponin level had declined to 0.63 ng / m on the 3rd day, and the patient was discharged with a referral for cardiac rehabilitation . We concluded on follow - up that otalgia in this patient was an atypical symptom of referred angina pain . The patient was seen 2 weeks later at the cardiology clinic of our hospital and reported no complaints about her condition or recurrence of ear pain . An ecg revealed no changes from the previous one done 2 weeks earlier at the time of discharge . The underlying pathophysiology of the referred otalgia in this patient can be explained by the autonomic dysfunction of the auricular branch of the vagus nerve often termed alderman s nerve . This branch of the vagus innervates the inner portion of the outer ear, and also controls the skeletal muscles including the superior, middle and inferior pharyngeal constrictors . The sinoatrial (sa) node we suggest that partial occlusion of the rca promoted damage of the parasympathetic fibers of the right vagus, and was the cause of referred otalgia and pharyngitis in our patient . Sensory nerves are affected by type 2 diabetes [47], but whether such a preexisting condition predisposes a person to angina - referred otalgia or pharyngitis is not clear . To our knowledge, symptoms of ear pain for cad and acute arterial occlusion in a patient have rarely been discussed in the literature . We found only one report; in this report the correct diagnosis of cardiac ischemia was missed in two patients who were initially treated for primary ear pain . In conclusion, clinicians should be aware and recognize that ear and throat pain may represent symptoms for cardiac ischemia.
Twenty healthy adults (twelve females and eight males) age 2252 years (average 34 years; without diabetes or known vascular disease) were studied supine or prone in a 7 t system (achieva, philips medical systems, cleveland, oh). Spectra were acquired with a partial - volume quadrature transmit / receive coil customized to fit the shape of a human calf . Axial, coronal, and sagittal t2-weighted turbo spin echo images were initially acquired of the left calf muscle . Typical parameters were: field of view 180 180 mm, repetition time (tr) 1,500 ms, echo time (te) 75 ms, turbo factor 16, and number of acquisitions (na), 1 . Single - voxel stimulated echo acquisition mode (steam) (typical parameters: voxel size 5 5 5 mm (0.1 ml), tr 2,000 ms, te 20 ms, spectral bw of 4 khz, number of points (np) 4,096 and zero - filled to 8,192 prior to fourier transform, na 16, no water suppression) was used to acquire h spectra from tibial bone marrow and subcutaneous fat tissue . To correct individual resonances for relaxation effects, t1 and t2 were measured in seven of the subjects . T1 was measured using inversion - recovery, with nine inversion delay times in the range of 5 ms to 3,000 ms, with tr 7 s and te 40 ms . T2 was measured by using ten te values from 20 ms to 180 ms, with tr 8 s. subjects were instructed to move slowly in the scan room . The entire scanning session was 60 min or less and it was well - tolerated by all subjects . All subjects were interviewed after the exam and again at 24 h after the exam . All subjects specifically denied dizziness, nausea, vertigo, headaches, or visual changes . (elysian, mn). To test the accuracy of the protocol to quantify lipid composition, phantom samples were prepared by mixing tristearin (18:0) (number of carbons: number of double bonds), triolein (18:1), and trilinolein (18:2) in the following ratios: 50:0:50, 50:10:40, 50:20:30, 50:30:20, 50:40:10, 50:50:0, and 25:50:25 . Phantom samples with composition of different chain lengths of triglyceride were prepared by mixing tripalmitin (16:0) and tristearin (18:0) in the following ratios: 100:0, 80:20, 75:25, 50:50, 25:75, 20:80 and 0:100 . All mixtures were dissolved in cd3cl in 4 ml glass vials, which were then mounted in the center of a 150 ml beaker filled with deionized water . Typical magnetic resonance spectroscopy (mrs) parameters were: tr 8 s, te 11 ms, voxel size 0.2 ml, np 16 k, bw 8 khz, na 128 . . The h chemical shift of in vivo fat resonances from bone marrow and subcutaneous tissue was assigned such that the methyl signal was at 0.9 ppm . Resonance areas were determined by fitting the spectra with voigt shapes (variable proportions of lorentzian plus gaussian) on acd software (advanced chemistry development, inc ., seven fatty acids predominate as follows (number of carbons: number of double bonds, typical abundance): myristic (14:0, 3%), palmitic (16:0, 1924%), palmitoleic (16:1, 67%), stearic (18:0, 36%), oleic (18:1, 4550%), linoleic (18:2, 1315%), and linolenic (18:3, 12%) (22, 23). These fatty acids account for well over 90% of the fatty acids in human adipose tissue . Odd - carbon fatty acids, longer chain fatty acids, and shorter chain fatty acids account for the remainder . Each of these less - abundant fats individually contributes much less than 1% (22). At 7 t, 10 resonances can be resolved, designated here as a to j in alphabetic order from upfield to downfield (fig . Six resonances contribute equivalent information about triglyceride composition: the ch3 methyl protons (labeled a, at 0.90 ppm), the ch2 methylene protons - (e, at 2.25 ppm) and - (c, at 1.59 ppm) to the carbonyl, and the glycerol backbone ch (i) and ch2 protons (g and h). Hence, there are only four additional informative resonances to consider: 1) bulk ch2 methylene protons (labeled b at 1.3 ppm); 2) allylic ch2 protons, - to a double bond, at 2.03 ppm (d); 3) diallylic (also called bis - allylic) ch2 protons at 2.77 ppm (f); and 4) olefinic, double bond -ch = ch- protons at 5.31 ppm (j), which partially overlap with the glycerol ch methine proton at 5.21 ppm (i). H nmr spectra of subcutaneous fat (left) and tibial bone marrow (right) from a 26 year - old healthy male at 7 tesla (7 t). The bottom trace shows the acquired spectrum, and the upper trace shows the fitted spectrum . A water signal is seen in the spectrum of subcutaneous fat but not bone marrow . A t2-weighted image shows the position of the voxel in the subcutaneous fat tissue and tibial bone marrow (5 5 5 mm) from which the spectra were acquired . It was assumed that the fatty acids detected here contain either 0, 1, or 2 double bonds . These three types of fatty acids account for 9798% of total fat in humans on ordinary western diets . Linolenic acid (18:3) is excluded in this simplification, but it contributes only 0.5% of the total triglycerides (22). With this assumption, fsat + fmono + fdi = 1 where fsat, fmono, and fdi refer to the fraction of fatty acids that are saturated, monounsaturated, and doubly unsaturated (or diunsaturated), respectively . The fraction that is diunsaturated, fdi, can be determined directly from the relative area of the resonance of the bridging diallylic protons (resonance f), with respect to the resonance of methylene protons to coo (resonance e):eq . 1\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation}{\mathrm{f}}_{{\mathrm{di}}}\;=\;{\mathrm{area}}\;({\mathrm{f / e}})\end{equation}\end{document} once the fdi value is determined, one can evaluate fmono from the relative area of proton resonance to the double bond by: eq . 2\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation}{\mathrm{f}}_{{\mathrm{mono}}}\;=\;0.5{\mathrm{area}}\;({\mathrm{d / e}})\;-\;{\mathrm{f}}_{{\mathrm{di}}}\end{equation}\end{document} the remaining unknown fsat, the fraction of saturated fatty acid, is derived as fsat = 1 (fmono + fdi). Assuming that f16c + f18c = 1, the fraction of fatty acids that are 16 carbon versus 18 carbon can be determined from the area of the bulk methylene resonances (-ch2-)n: eq . 3\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation}{\mathrm{area}}\;({\mathrm{b / e}})\;=\;{\mathrm{f}}_{{\mathrm{16c}}}(12{\mathrm{f}}_{{\mathrm{sat}}}\;+\;8{\mathrm{f}}_{{\mathrm{mono}}})\;+\;{\mathrm{f}}_{{\mathrm{18c}}}(14{\mathrm{f}}_{{\mathrm{sat}}}\;+\;10{\mathrm{f}}_{{\mathrm{mono}}}\;+\;7{\mathrm{f}}_{{\mathrm{di}}})\end{equation}\end{document} the coefficients in front of the individual fractions are: 12 for palmitic acid (16:0), 8 for palmitoleic acid (16:1), 14 for stearic acid (18:0), 10 for oleic acid (18:1), and 7 for linoleic acid (18:2). This analysis is essentially identical to the earlier analysis (20) with the exception that a term for an unsaturated fat with three double bonds was omitted rather than assuming a low, fixed concentration . Twenty healthy adults (twelve females and eight males) age 2252 years (average 34 years; without diabetes or known vascular disease) were studied supine or prone in a 7 t system (achieva, philips medical systems, cleveland, oh). Spectra were acquired with a partial - volume quadrature transmit / receive coil customized to fit the shape of a human calf . Axial, coronal, and sagittal t2-weighted turbo spin echo images were initially acquired of the left calf muscle . Typical parameters were: field of view 180 180 mm, repetition time (tr) 1,500 ms, echo time (te) 75 ms, turbo factor 16, and number of acquisitions (na), 1 . Single - voxel stimulated echo acquisition mode (steam) (typical parameters: voxel size 5 5 5 mm (0.1 ml), tr 2,000 ms, te 20 ms, spectral bw of 4 khz, number of points (np) 4,096 and zero - filled to 8,192 prior to fourier transform, na 16, no water suppression) was used to acquire h spectra from tibial bone marrow and subcutaneous fat tissue . To correct individual resonances for relaxation effects, t1 and t2 were measured in seven of the subjects . T1 was measured using inversion - recovery, with nine inversion delay times in the range of 5 ms to 3,000 ms, with tr 7 s and te 40 ms . T2 was measured by using ten te values from 20 ms to 180 ms, with tr 8 s. subjects were instructed to move slowly in the scan room . The entire scanning session was 60 min or less and it was well - tolerated by all subjects . All subjects were interviewed after the exam and again at 24 h after the exam . All subjects specifically denied dizziness, nausea, vertigo, headaches, or visual changes . (elysian, mn). To test the accuracy of the protocol to quantify lipid composition, phantom samples were prepared by mixing tristearin (18:0) (number of carbons: number of double bonds), triolein (18:1), and trilinolein (18:2) in the following ratios: 50:0:50, 50:10:40, 50:20:30, 50:30:20, 50:40:10, 50:50:0, and 25:50:25 . Phantom samples with composition of different chain lengths of triglyceride were prepared by mixing tripalmitin (16:0) and tristearin (18:0) in the following ratios: 100:0, 80:20, 75:25, 50:50, 25:75, 20:80 and 0:100 . All mixtures were dissolved in cd3cl in 4 ml glass vials, which were then mounted in the center of a 150 ml beaker filled with deionized water . Typical magnetic resonance spectroscopy (mrs) parameters were: tr 8 s, te 11 ms, voxel size 0.2 ml, np 16 k, bw 8 khz, na 128 . The h chemical shift of in vivo fat resonances from bone marrow and subcutaneous tissue was assigned such that the methyl signal was at 0.9 ppm . Resonance areas were determined by fitting the spectra with voigt shapes (variable proportions of lorentzian plus gaussian) on acd software (advanced chemistry development, inc ., human adipose tissue is composed largely of triglycerides . Seven fatty acids predominate as follows (number of carbons: number of double bonds, typical abundance): myristic (14:0, 3%), palmitic (16:0, 1924%), palmitoleic (16:1, 67%), stearic (18:0, 36%), oleic (18:1, 4550%), linoleic (18:2, 1315%), and linolenic (18:3, 12%) (22, 23). These fatty acids account for well over 90% of the fatty acids in human adipose tissue . Odd - carbon fatty acids, longer chain fatty acids, and shorter chain fatty acids account for the remainder . Each of these less - abundant fats individually contributes much less than 1% (22). At 7 t, 10 resonances can be resolved, designated here as a to j in alphabetic order from upfield to downfield (fig . 1). Six resonances contribute equivalent information about triglyceride composition: the ch3 methyl protons (labeled a, at 0.90 ppm), the ch2 methylene protons - (e, at 2.25 ppm) and - (c, at 1.59 ppm) to the carbonyl, and the glycerol backbone ch (i) and ch2 protons (g and h). Hence, there are only four additional informative resonances to consider: 1) bulk ch2 methylene protons (labeled b at 1.3 ppm); 2) allylic ch2 protons, - to a double bond, at 2.03 ppm (d); 3) diallylic (also called bis - allylic) ch2 protons at 2.77 ppm (f); and 4) olefinic, double bond -ch = ch- protons at 5.31 ppm (j), which partially overlap with the glycerol ch methine proton at 5.21 ppm (i). H nmr spectra of subcutaneous fat (left) and tibial bone marrow (right) from a 26 year - old healthy male at 7 tesla (7 t). The bottom trace shows the acquired spectrum, and the upper trace shows the fitted spectrum . A water signal is seen in the spectrum of subcutaneous fat but not bone marrow . A t2-weighted image shows the position of the voxel in the subcutaneous fat tissue and tibial bone marrow (5 5 5 mm) from which the spectra were acquired . It was assumed that the fatty acids detected here contain either 0, 1, or 2 double bonds . These three types of fatty acids account for 9798% of total fat in humans on ordinary western diets . Linolenic acid (18:3) is excluded in this simplification, but it contributes only 0.5% of the total triglycerides (22). With this assumption, fsat + fmono + fdi = 1 where fsat, fmono, and fdi refer to the fraction of fatty acids that are saturated, monounsaturated, and doubly unsaturated (or diunsaturated), respectively . The fraction that is diunsaturated, fdi, can be determined directly from the relative area of the resonance of the bridging diallylic protons (resonance f), with respect to the resonance of methylene protons to coo (resonance e):eq . 1\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation}{\mathrm{f}}_{{\mathrm{di}}}\;=\;{\mathrm{area}}\;({\mathrm{f / e}})\end{equation}\end{document} once the fdi value is determined, one can evaluate fmono from the relative area of proton resonance to the double bond by: eq . 2\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation}{\mathrm{f}}_{{\mathrm{mono}}}\;=\;0.5{\mathrm{area}}\;({\mathrm{d / e}})\;-\;{\mathrm{f}}_{{\mathrm{di}}}\end{equation}\end{document} the remaining unknown fsat, the fraction of saturated fatty acid, is derived as fsat = 1 (fmono + fdi). Assuming that f16c + f18c = 1, the fraction of fatty acids that are 16 carbon versus 18 carbon can be determined from the area of the bulk methylene resonances (-ch2-)n: eq . 3\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation}{\mathrm{area}}\;({\mathrm{b / e}})\;=\;{\mathrm{f}}_{{\mathrm{16c}}}(12{\mathrm{f}}_{{\mathrm{sat}}}\;+\;8{\mathrm{f}}_{{\mathrm{mono}}})\;+\;{\mathrm{f}}_{{\mathrm{18c}}}(14{\mathrm{f}}_{{\mathrm{sat}}}\;+\;10{\mathrm{f}}_{{\mathrm{mono}}}\;+\;7{\mathrm{f}}_{{\mathrm{di}}})\end{equation}\end{document} the coefficients in front of the individual fractions are: 12 for palmitic acid (16:0), 8 for palmitoleic acid (16:1), 14 for stearic acid (18:0), 10 for oleic acid (18:1), and 7 for linoleic acid (18:2). This analysis is essentially identical to the earlier analysis (20) with the exception that a term for an unsaturated fat with three double bonds was omitted rather than assuming a low, fixed concentration . Ten lipid resonances are typically observed in the 7 t h spectrum from physiological fats (fig . 1). Except for the double - bond protons, which partially overlap the methine proton of the glycerol backbone (the chemical shift difference is about 0.1 ppm), the lipid resonances were well - resolved at 7 t and qualitatively similar to high - resolution spectra obtained in mice (20). The water signal generally appears in the h spectrum of subcutaneous fat tissue, with subject - dependent intensity and line width, but it is nearly undetectable in bone marrow acquired from a small voxel (0.1 ml). The resonance assignments, chemical shifts, and relative intensities from both marrow and subcutaneous fat are summarized in table 1 . Chemical shifts and relative resonance areas the areas are relative to the methylene resonance to coo (peak e) after correction for partial saturation . The validity of this analysis was tested by mixing three c18 triacylglycerols and, in a separate experiment, by mixing c16 and c18 triacylglycerols, in various ratios . Figure 2a shows data collected from phantoms, with the area (f / e) plotted against the actual known fraction of trilinolein (fdi) in the c18 mixture phantom samples . A linear correlation is seen between the measured f / e ratio and the sample true fdi value, with linear coefficient of 1.02 and correlation coefficient r = 0.992 . Figure 2b shows the measured c18 triacylglycerol faction (f18c) against the actual known c18 fraction in the c16 and c18 mixture phantom samples . The plot of the known f18c versus the measured f18c, which was evaluated by 0.5 * area (b / e) 12, also yielded a linear dependence, with linear coefficient of 0.94 and correlation coefficient r = 0.992 . A: correlation of the known, prepared fraction of diunsaturated triglyceride with mr - measured fraction of diunsaturated triglyceride . Phantoms were prepared by mixing three c18 triglycerides (number of double bonds), tristearin (0), triolein (1), and trilinolein (2) in the following ratios (tristearin: triolein: trilinolein): 50:0:50, 50:10:40, 50:20:30, 50:30:20, 50:40:10, 50:50:0, and 25:50:25 . B: correlation of the known, prepared fraction of c18 triglyceride with mr - measured value from area (b / e). Phantoms were prepared by mixing c16 tripalmitin and c18 tristearin with the following ratios (c16:c18): 100:0, 80:20, 75:25, 50:50, 25:75, 20:80, and 0:100 . The data were analyzed using f18c = 0.5 * area (b / e) 12 . For quantitation of fat composition in human subcutaneous tissue and tibial marrow, the signal intensities were corrected for all differences in t1 and t2 as determined from inversion - recovery (fig . Figure 3 shows the t1 (left) and t2 (right) spectra collected from the same voxel located in subcutaneous tissue, together with the corresponding curve fittings (bottom). Figure 4 compares the t2 data between tibial marrow and subcutaneous tissue collected from same - sized voxels, on the same volunteer . The measured t1 and t2 values at this field are summarized in table 2 . As shown by the data, protons at different structural positions in fats have quite different values, with t1s ranging from 0.321.16 s, and t2s ranging from 3074 ms . Calf subcutaneous fat has shorter t1 and t2 values, about 7% on average, in comparison with tibial bone marrow . It should be pointed out that because of the presence of proton j - coupling, these t1 and t2 values are valid only for steam sequence . Shorter t2 values have been reported for animal abdominal fat using point - resolved spectroscopy (press) sequence (20) at the same field strength . Inversion - recovery measurement of t1 (left panel) and echo time (te)-dependence measurement of t2 (right panel) from subcutaneous fat tissues of a 34 year - old healthy male at 7 t . The inversion bandwidth (bw) was set to span resonances a and b (middle stack), or resonances c, d, e, and f (left - most stack). The inversion delay times for the given spectra are 10, 20, 50, 100, 200, 500, 1,000, 2,000, and 3,000 ms with a constant repetition time (tr) of 7 s and te of 40 ms (left panel). The echo times for the t2 measurements are 20, 40, 60, 80, 100, 140, and 180 ms (right panel). T2 measurement of tibia bone marrow (left panel) and calf subcutaneous fat (right panel) from a 25 year - old healthy female by varying tes at 7 t . Note that the voxel (5 5 5 mm) fits well in the single fat cell of the subcutaneous tissue and the collected h spectrum is water - free . All spectra are vertically scaled to equal magnitude of the methylene resonance (b), and as a result, with te increase, the methyl resonance a with longer t2 than b shows signal rising, whereas the shorter t2 resonances such as c and d show signal decaying relative to resonance b. to avoid overcrowding, the fitting of the a and c peaks is not shown . Other parameters: tr 8 s, number of acquisitions, 8; number of points, 4 k; bw 4 khz . N = 36) the values shown are mean sd and are valid for the stimulated echo acquisition mode pulse sequence used . The derived fractions of saturated, monounsaturated and diunsaturated fat constituents are summarized in table 3, together with the estimated fraction of fatty acid chain length f16c and f18c . The composition of bone marrow and adipose fat was not significantly different, with an average 29.1 3.5% saturated, 46.4 4.8% monounsaturated, and 24.5 3.1% diunsaturated fractions for marrow, as compared with 27.1 4.2% saturated, 49.6 5.7% monounsaturated, and 23.4 3.9% diunsaturated fractions for subcutaneous fat . A good linear correlation was seen between tibial marrow and subcutaneous fat in the measured area ratio (f / e), which is the index of diunsaturated fraction, for the 20 subjects studied, as shown in fig ., a composition of 33.4 4.9% from fatty acid with 16 carbons and 66.5 9.7% from fatty acid with 18 carbons was calculated for bone marrow, as compared with subcutaneous fat with a composition of 23.4 3.9% for 16 carbon fraction and 73.8 17.8% for 18 carbon fraction . Average fat composition in marrow and subcutaneous tissues the relative concentration, as percentage of saturated, monounsaturated, and diunsaturated fats, as well as the fraction of 16- vs. 18-carbon fats are shown . Correlation of measured area (f / e) between tibial bone marrow and subcutaneous fat for the 20 healthy adult subjects studied, showing that the diunsaturated fatty acid is similar for these two adipose sites and that the fat composition variation among subjects is detectable by h magnetic resonance spectroscopy . High - quality h nmr spectra from human adipose tissue were obtained routinely at 7 t . The features of the h spectra at 7 t were consistent with spectra of triacylglycerols obtained under high - resolution conditions (812) or in mice at 7 t (20). Perhaps the most significant observation was that chemical shift dispersion in humans is greatly improved compared with 1.5 t or 3.0 t. the ability to resolve protons adjacent to double bonds allows noninvasive estimation of the fatty acid composition of adipose tissue . Proton - decoupled c nmr of human adipose tissue (1519) allows more - detailed characterization of fat composition, compared with a h spectrum . For example, the outer unsaturated carbons in a bisallylic group (-ch2-ch = ch - ch2-ch = ch - ch2-) are resolved from the inner carbons in spectra obtained in vivo at 1.5 t. natural abundance c nmr is limited by relatively low sensitivity, low spatial resolution and additional technical requirements, all disadvantages compared with h spectroscopy for human study . Conversely, 7 t instruments are not widely available . However, because the number of 7 t instruments in medical sites is increasing steadily and h spectroscopy is routine on all systems, it seems reasonable to anticipate use of h spectroscopy at 7 t for clinical research . At 7 t the three resonances in the narrow range of 2.032.77 ppm provide an internal standard plus information about the abundance of protons between or adjacent to double bonds . This information, after correction for relaxation, allows calculation of the relative concentration of saturated, monounsaturated, and diunsaturated fatty acids . The fraction of saturated fats in triglycerides as determined by h nmr spectroscopy at 7 t, about 2729%, is in excellent agreement with literature values . However, the fraction of fatty acids that are monounsaturated was somewhat less (about 4650%) in this study, compared with biopsy studies, where about 5560% monounsaturated fatty acids are typically found (22, 24). Consequently, the fraction of fatty acids estimated to be diunsaturated was about 2324% in this study compared with about 1718% in biopsy studies . First, macromolecules or aqueous metabolites with chemical shifts overlapping the lipid resonances may alter the estimates of relative intensity . Second, the signals in vivo are the sum of very complex h spin - coupled multiplets from several different fatty acids with slightly different chemical shifts . Consequently, a single lorentzian - gaussian line does not properly represent the observed lineshape . Even when high - resolution analytical h nmr was used to measure marrow fat composition from samples extracted in chloroform, thereby removing aqueous species and other complicating factors that may contribute error in the study of intact tissue, the nmr method overestimated the fraction of saturated fatty acids, compared with gold standard third, as emphasized earlier (20), correction for t2 is also essential and more important than correction for t1 differences . For a typical te of 11 ms, the relative difference in t2 correction among the resonances d, e, and f accounts for 4%, but it reaches 8% at te = 20 ms, and is as large as 18% at te = 40 ms . This compares to only about 1% difference after correction for t1 effects for the same three resonances at tr = 2 s. the t1 correction is not needed when the spectrum is collected with tr of 4 s. it is found that the calf subcutaneous fat has relatively shorter (7%) proton relaxation times than the marrow (table 2), indicating a larger local bo field inhomogeneity on subcutaneous tissue . This can be understood from the difference in the microscopic structure between these two types of adipose tissues, as shown in the mri images (fig . 4, insert), in which subcutaneous tissue is seen as packed with large fat cells of different sizes, embedded with rich vasculature, and curvedly shaped, whereas the bone marrow appears as fine uniform structure, aligned straight inside the tibial bone, and nearly parallel to the bo field . Because of this, the spectral resolution from bone marrow is generally superior to that from subcutaneous fat, as shown in fig . It is probably unrealistic to anticipate perfect correlation between this mr analysis of extremity fat and marrow compared with literature data obtained largely from the abdomen, chest, or buttocks . There are complex effects of diet, season, gender, and anatomical site on fatty acid composition (24, 25). For example, extremity adipose tissue has a greater fraction of monounsaturated fat and less saturated fat compared with subcutaneous fat from the chest and abdomen (25). Nevertheless, the average ratio fdi / fmono (47% for subcutaneous fat and 53% for bone marrow, table 3) obtained from this h mrs study for 20 adult subjects (average age 34), is in excellent agreement with a previously reported value (50%) for the ratio of polyunsaturated to monounsaturated fatty acid by in vivo c study (19) of calf subcutaneous tissue of 17 adult subjects (average age 30). The obvious advantage of h mrs is that it is fast (less than 1 min), has a low specific absorption rate (no need for broadband decoupling as in c), is easily localized (not a volume detection), and is typically more easily implemented (no special scanner hardware or coil required). The high resolution and sensitivity of the current steam - based h mr analysis also enable the detection of fat composition variation among uncontrolled healthy subjects (fig . 5), which might be attributed to the difference in individuals long - term diet and metabolic genetics . Therefore, it is anticipated that the method may be useful in rapid detection of substantial changes in the composition of fatty acids in response to diet, exercise, and fat - metabolic diseases . This method, in our view, would help advance noninvasive human lipid research, which has been focused on studying fat mass distribution by mri and other techniques rather than fat molecular structural composition . In addition, an analysis of our data (not shown) indicates no obvious difference in fat composition between male and female subjects, although, on average, the calf subcutaneous fat in female is approximately two - fold thicker than in male . The decision to simplify the description of fatty acid composition to saturated, monounsaturated, and diunsaturated fats was based on two considerations . First, the amount of polyunsaturated fats (three or more double bonds) in human adipose tissue contributes very little, less than 12%, to the total h nmr signal . Second, earlier studies that were able to quantify triply unsaturated fatty acids separate from other fatty acids required high - resolution analytical conditions (1012). For practical purposes in humans, this achievement would mean the capacity to separately quantify oleic (18:1), linoleic (18:2), and linolenic (18:3) acid . However, at the resolution achieved in these studies, a 50:50 mixture of triolein: trilinolenin is indistinguishable from a spectrum of trilinolein . For these reasons, the description of fatty acids as saturated, monounsaturated, or diunsaturated seems a reasonable simplification . Frequency - dependent spatial localization introduces a chemical shift displacement artifact in the h mr spectrum . Quantitation may be inaccurate if it is based on ratios of two resonances when the region of interest is close to the inter - tissue boundary . Although this effect was not critically evaluated in this study, the use of a small voxel (0.1 ml) well within the bone marrow plus analysis of resonances over a narrow chemical shift range reduces susceptibility to chemical shift displacement effects . Unlike the methyl resonance, the resonance of protons to the carbonyl is not affected by the neighboring large bulk ch2 (resonance b), which often induces an uneven baseline at nearby resonances at shorter tes . Although this baseline problem can be improved at longer tes, the faster decay of protons to a double bond (resonance d, t2 = 42 ms for marrow, and 39 for subcutaneous fat) than the reference resonance e (t2 = 59 ms) makes long te scan conditions less favorable . Steam sequence has the advantage of shorter te than press and less dependence on proton j modulation due to the smaller te / t2 ratio, and therefore was chosen for the quantification of fat composition in this method . In addition to composition analysis, the well - resolved resonances of human tibial bone marrow and calf subcutaneous fat at 7 t could also be very useful as an internal standard to quantify intramuscular lipids (26), which have attracted great research interest over the last few years, owing to their relation to insulin sensitivity, diabetes, and obesity (27). In conclusion, this h mrs study at 7 t indicates that the rapid acquisition of high - quality h nmr spectra from subcutaneous fat and from bone marrow is straightforward in healthy volunteers, and that the spectra may be analyzed to assess triglyceride composition . This may facilitate longitudinal monitoring of changes in lipid composition in response to diet, exercise, and disease.
In canada, fish contribute to over 50% of the dietary intake of mercury (hg) by adults (dabeka et al . 2003), and fish containing the highest concentrations of hg are predatory fish, such as shark, swordfish and tuna (health canada 2007). Hg, present in fish as both methyl - hg and inorganic hg, is a potent neurotoxicant . On the other hand, fish are one of the best food sources of vitamin d and omega-3 fatty acids, dha and epa, and it is important to weigh the benefits of eating fish against the potential toxicity of hg present in the fish (health canada 2007). To promote consumption of fish while at the same time providing advice to vulnerable population groups about limiting consumption of predatory fish, and to evaluate the risk - to - benefit ratio of eating fish, it is important to know the levels of hg in predatory fish sold at the retail level in canada . In 2002, 2004) which found that predatory fish, such as shark, swordfish and tuna, contained high concentrations of hg, averaging 1820 ng g for shark, 1430 ng g for marlin and 930 ng g for fresh or frozen tuna . This survey is a supplement to the previous work and provides levels of hg found in retail fish purchased in 2005 . Marlin, sea bass, red snapper, orange roughy, fresh water trout, grouper, black cod (sablefish), arctic char, king fish (king mackerel) and tilefish are included in this survey ., samples were purchased as cleaned fillets or whole fish at the retail level in toronto, vancouver and montreal in 2005 . The retail stores included four supermarket chains and four specialty fish seafood outlets in each city . Each sample was shipped frozen to maxxam analytics inc . For cleaning (if necessary), homogenization and bottling in pre - cleaned polyethylene bottles supplied for total hg and glass jars with teflon cap liners for methyl hg (i - chem brand, thermo fisher scientific). The samples were then frozen and shipped to health canada's food research division in ottawa for analysis . Information provided with each sample included, where available, the species name as provided by the retail outlet, the state of the sample when purchased (frozen, fresh or previously frozen), the city of sample pickup, name of store where purchased, country of origin and date of collection . The countries of origin for the samples were usa (7), argentina (1), australia (5), canada (26), chile (13), hungary (2), mexico (1), new zealand (3) and taiwan (1), with the remaining samples being of unknown origin . Analyses were performed using the reagents, instrumentation and methodology described in dabeka et al . Briefly, after a low - temperature nitric / hydrochloric acid and hydrogen peroxide digestion of roughly 1 g fish tissue and dilution to 50 ml with water, measurements were made using a cetac-6000a dedicated hg analyser equipped with an asx-500 autosampler and adx autodilutor . Quality control measures for each analytical batch included three reagent blanks, two reagent blank spikes (200 ng hg), one sample spike of 400 ng hg (in duplicate for both the unspiked and spiked 400 ng sample) and duplicates of three different standard reference materials (srms) with certified hg concentrations . Two cross - check standards (from a different manufacturer) were included in each batch for standard verification during the run . The solution limit of detection (lod) was estimated for each analytical batch by multiplying the standard deviation of the three reagent blanks by 3 . Sample lods were calculated by multiplying the solution lod by the dilution volume and dividing by the weight of the actual sample taken for analysis . Solution lods averaged 0.045 ng ml and sample lods averaged 1.9 ng g. recoveries from spiked blanks and samples averaged 97 and 101%, respectively . One of the sample spike recoveries was high (155% in batch 8), and this was due to the high concentration of hg in the sample selected for spiking in the batch compared with the spike added . Agreement with certified levels in the national research council of canada (nrcc) dolt-2 and dorm-2 srms and the national institute of standards and technology (nist) oyster tissue 1566a srm was generally satisfactory, although results for batches 9, 10 and 11 were unusually high . Blank spaces in the tables denote that the particular test was not included in the batch . Note: blank spaces denote that the particular test was not included in the batch ., samples were purchased as cleaned fillets or whole fish at the retail level in toronto, vancouver and montreal in 2005 . The retail stores included four supermarket chains and four specialty fish seafood outlets in each city . Each sample was shipped frozen to maxxam analytics inc . For cleaning (if necessary), homogenization and bottling in pre - cleaned polyethylene bottles supplied for total hg and glass jars with teflon cap liners for methyl hg (i - chem brand, thermo fisher scientific). The samples were then frozen and shipped to health canada's food research division in ottawa for analysis . Information provided with each sample included, where available, the species name as provided by the retail outlet, the state of the sample when purchased (frozen, fresh or previously frozen), the city of sample pickup, name of store where purchased, country of origin and date of collection . The countries of origin for the samples were usa (7), argentina (1), australia (5), canada (26), chile (13), hungary (2), mexico (1), new zealand (3) and taiwan (1), with the remaining samples being of unknown origin . Analyses were performed using the reagents, instrumentation and methodology described in dabeka et al . (2002). Briefly, after a low - temperature nitric / hydrochloric acid and hydrogen peroxide digestion of roughly 1 g fish tissue and dilution to 50 ml with water, measurements were made using a cetac-6000a dedicated hg analyser equipped with an asx-500 autosampler and adx autodilutor . Quality control measures for each analytical batch included three reagent blanks, two reagent blank spikes (200 ng hg), one sample spike of 400 ng hg (in duplicate for both the unspiked and spiked 400 ng sample) and duplicates of three different standard reference materials (srms) with certified hg concentrations . Two cross - check standards (from a different manufacturer) were included in each batch for standard verification during the run . The solution limit of detection (lod) was estimated for each analytical batch by multiplying the standard deviation of the three reagent blanks by 3 . Sample lods were calculated by multiplying the solution lod by the dilution volume and dividing by the weight of the actual sample taken for analysis . Solution lods averaged 0.045 ng ml and sample lods averaged 1.9 ng g. recoveries from spiked blanks and samples averaged 97 and 101%, respectively . One of the sample spike recoveries was high (155% in batch 8), and this was due to the high concentration of hg in the sample selected for spiking in the batch compared with the spike added . Agreement with certified levels in the national research council of canada (nrcc) dolt-2 and dorm-2 srms and the national institute of standards and technology (nist) oyster tissue 1566a srm was generally satisfactory, although results for batches 9, 10 and 11 were unusually high . Blank spaces in the tables denote that the particular test was not included in the batch . Note: blank spaces denote that the particular test was not included in the batch . A summary of the results (table 2) found that arctic char, red snapper and fresh water trout contained the lowest concentrations of hg, averaging 37, 148 and 55 ng g, respectively . Average levels in the other fish types varied from 384 ng g for black cod to 854 ng g for marlin . Note: designated fish subject to a 1000 ngg regulatory limit, others to a limit of 500 ngg average concentrations of hg in the different species were in general agreement with those found in other canadian and international surveys (table 3). Differences among the surveys can be attributed to the size of the individual fish from which the sample was taken, as fish mercury concentrations vary directly with fish size . Additionally, regional differences in hg concentrations may be a function of the food source . For example, tilefish caught in the atlantic ocean contained 144 ng g hg, whereas those sampled in the gulf of mexico averaged 1450 ng g (fda 2009). It is uncertain whether this difference is due to different food sources or to the situation that the tilefish in the two areas are different species . Comparison of hg levels (ngg) found in this study with those found previously in canada and other countries . Average of three tilefish - two samples purchased in 2008 containing 128 and 3000 ng g, and one purchased in 2005 contained 80 ng g. fda (2009); environmental protection agency (2000); national marine fisheries service (1978). The primary reference for the gulf tilefish and sablefish data in the fda report is a national marine fisheries service report (hall et al . 1978), while that for the kingfish or king mackerel is an epa report (ache et al . 144 ng g in atlantic tilefish and 1450 ng g in those caught in the gulf of mexico (fda 2009). The current canadian regulatory limit for total mercury in the edible portion of commercially sold fish is 500 ng g, with the exception of a 1000 ng g limit and accompanying consumption advice (health canada, 2008) for specific species of piscivorous fish: shark, swordfish, marlin, orange roughy, escolar, and fresh and frozen tuna (health canada, 2010). The proportion of individual samples exceeding the relevant standards was: 13% of black cod, 24% of grouper, 40% of king fish, 28% of marlin, 20% of sea bass and 50%) of tile fish (table 2). However, the uncertainty in these percentages is high due to the small numbers of each species tested . The mean and median concentrations of mercury in black cod, grouper, king fish, marlin and sea bass were all below the standards for the respective species . Additional data would aid in characterizing the typical mercury concentrations in some types of fish, in particular king fish and tilefish . For example, the us fda has reported a median mercury value in tilefish (atlantic) of only 99 ng g (n = 32) (fda, 2009). It should also be noted that the occasional consumption of infrequently consumed fish containing mercury levels greater than the regulatory limits would not be expected to pose a health risk to consumers . Thanks are due to elizabeth elliott and mark feeley of the chemical health hazard assessment division, bureau of chemical safety for review and helpful suggestions.
Surprisingly, during summer and autumn 2014, sbv reappeared in germany to a greater extent . Several samples from various federal states were submitted to the friedrich - loeffler - institut, federal research institute for animal health (insel riems, germany), to confirm an sbv infection (table) and to further characterize these reemerging viruses . To evaluate sequence variations among sbv variants circulating in germany since 2011, the original sbv isolate (bh80/11) and viruses isolated in 2012 from the blood of viremic sheep (bh619/12) or cattle (d495/12 - 1 and bh652/12) were compared with 3 genomes obtained from the viruses in acutely infected adult cattle in 2014 (bh119/14 - 1/2, bh119/14 - 3/4, bh132/14). Rna was extracted by using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer s recommendations, and the open reading frames of all 3 genome segments (large [l], medium [m], and small [s] segment; primer sequences available on request) were sequenced as described (7). Kp731865kp731882). Sequence alignments and translation in amino acids were supported by geneious version 7.1 (biomatters, auckland, new zealand). We generated a maximum - likelihood tree (hasegawa - kishino - yano model, 1,000 bootstrap replicates) using mega5 (8). For the phylogenetic analysis of the m segment, sequences previously obtained from organ samples from newborns malformed because of sbv infection also were integrated (7). For the s segment (830-nt long), which encodes the nucleocapsid protein (n) and a nonstructural protein (nss), the samples obtained from acutely infected animals in 2014, bh619/12 and bh652/12, were 100% identical to the original sbv strain bh80/11 from 2011 . In the sample d495/12 - 1 from 2012, a single nucleotide was substituted (technical appendix figure). The viral rna - dependent rna polymerase encoding l segment (6864 nt) also showed high stability, and only a few nucleotide substitutions compared with bh80/11 were found in the latest samples (d495/12 - 1 and bh652/12: 6 nt; bh619/12: 10 nt; bh119/14 - 1/2, bh119/14 - 3/4 . And bh132/14: 18 nt). Overall, sequence identity was 99.7%99.9% (technical appendix figure). The m segment (4415-nt long) encodes 2 glycoproteins (gn and gc) and a nonstructural protein (nsm). It is the most variable genome segment of sbv and related viruses (7,9,10). Despite the identification of a highly variable region within the gc - coding sequence in viruses in malformed newborns (7,9), there was a high sequence stability of the viruses detected in the blood of acutely infected adult animals; all sequences clustered closely (figure 1). In contrast to the maximum of 77 nt and 43 aa substitutions or 12 aa deletions or 2 aa insertions found in organ samples of lambs or calves (7), we detected only 612 nt and 2 aa (bh619/12), 3 aa (d495/12 - 1, bh119/14 - 3/4, bh132/14) or 4 aa (bh652/12, bh119/14 - 1/2) substitutions (technical appendix figure). Because gn and gc are major immunogens of orthobunyaviruses (10), the mutation hot spot was supposed to be involved in immune evasion mechanisms and/or adaption of the cell tropism within the individual host (7,9). However, insect - transmitted viruses such as sbv have to adapt to 2 hosts and undergo replication cycles in both the arthropod vector and the mammalian host . Thus, the high sequence stability of virus strains detectable in viremic animals might be necessary for transmission to the vector . Notably, in comparison with the original sbv isolate, ke substitutions at aa 746 and 1340 of the m segment were found in all other samples . Because these mutations have now been consistently present for at least 2 years, they might have occurred during the adaptation to european ruminants or insects and could provide a growth advantage within the individual host or might be beneficial for transmission between host and vector . Phylogenetic analysis based on nucleotide sequences of the medium segment of schmallenberg virus samples isolated from blood of acutely infected animals in 2011 or 2012 (blue) or sequenced directly from the blood of viremic cattle in 2014 (red) and from organ samples of malformed newborns (black) (7). Scale bar indicates nucleotide substitutions per site . To investigate whether the detected sequence variations correlated with any change in pathogenicity, 5 female sheep and 1 female calf were subcutaneously inoculated with pools of up to 5 serum or whole blood samples from 1 of the holdings with new cases confirmed in 2014 (sheep 1 and 3 and the calf with samples from lower saxony; sheep 2, 4, and 5 with samples from saxony [permit no . Blood samples were taken daily for the first 2 weeks after inoculation and analyzed by real - time reverse transcription pcr (11). Thereafter, serum samples were taken in weekly intervals and tested in a microneutralization assay (12) and a commercially available sbv antibody elisa (i d screen schmallenberg virus competition; idvet, grabels, france). The calf and sheep 25 became infected; viral genome was detectable in their blood for 46 days (figure 2), which agrees with the short - lived viremia previously observed after experimental infection of cattle and sheep with the original sbv sample or the first cell culture isolate (1,13). Antibodies were first detected on day 7 after infection (sheep 2, sheep 5) or day 14 after infection (calf, sheep 3, sheep 4) in both tests . Detection of schmallenberg virus genome in the blood of experimentally infected cattle and sheep, germany, 2014 . Virus genome with a high sequence identity to the first sbv sample was repeatedly detected in the blood of acutely infected adult cattle, and in experimentally infected animals, viremia developed that was identical to the original sbv isolate . The renewed virus circulation during the 2014 vector season was observed primarily in an area less affected in the 2 previous years . The missing or markedly reduced virus circulation led to a decline in herd seroprevalence caused by a missing infection of the young stock (14,15); further reasons for the unexpected recurrence of sbv could be persistence within the insect vectors . As a consequence, the infection of naive animals in autumn 2014 resulted in an increasing frequency of the birth of malformed offspring in the following winter . Comparison of the small-, medium-, and large - segment sequences of schmallenberg viruses isolated in 2012 from the blood of viremic ruminants or detected in 2014 in acutely infected adult cattle with the first sbv isolate from 2011, germany.
Intracranial foreign bodies frequently result from trauma, including penetrating injury, and rarely involve wooden materials compared with metallic materials . When intracranial penetrations of wooden objects occur through the transnasal or transorbital route, physical examinations may reveal no abnormalities, and the objects may be difficult to visualize with conventional radiography . We present a case involving the transnasal intracranial penetration of a wooden branch that resulted in a delayed intracranial infection due to initial misdiagnosis . A 48-year - old man presented to the emergency department with complaints of headache the day before . Two days earlier, he fell to the ground in a drunken state, and he could not clearly remember the incident . At the emergency department, no neurological or physical abnormalities, such as external contusions or cerebrospinal fluid rhinorrhea, were observed . Computed tomography (ct) that was performed upon admission showed a round hypodense signal in the left frontal area, which suggested pneumocephalus, and a small amount acute subdural hematoma in the left frontotemporoparietal area (figure 2 and 3). The patient was admitted to the neurosurgical department for close observation, and prophylactic antibiotics were administered for the pneumocephalus . Brain magnetic resonance imaging (mri) revealed a fistula from the left nasal cavity to the frontal lobe with enhancement along the tract and ventricular lining (figure 4). The foreign body was a wooden branch (11 cm long and 0.7 cm wide) covered with brain tissue (figure 5). After the operation, the patient received intravenous antibiotics (ceftriaxone plus vancomycin) for over 2 months . A microbiologic culture study on the foreign body showed gram - positive cocci (s. aureus). Intracranial penetrating injuries by foreign bodies represent only 0.4% of all head injuries and usually occur in children because of falls.17) intracranial foreign bodies that cause severe intracranial infections can be fatal.8) miller et al.7) reviewed 42 cases with intracranial penetrating injuries and found that 64% developed central nervous system infections, 45% had additional brain abscesses, and the death rate was 25% . Generally, the time from the injury to the clinical intracranial infection presentation is long, but it can vary from a few days to several years.5) therefore, careful history taking and thorough physical and radiologic examinations are essential . However, intracranial wooden foreign bodies are difficult to detect with plain radiographic techniques and distinguish from periorbital fat and air in the nasal cavity on ct images.48) the ct attenuation values of wooden foreign bodies vary depending on their water content . Freshly cut wood has a relatively high physical density because of its high water content; therefore, it is difficult to distinguish it from soft tissues, such as muscle and vitreous.3) conversely, when wood dries, the water is gradually replaced by gas, and its density is difficult to distinguish from that of fat or air.3) consequently, intracranial wooden foreign bodies have variable densities over time and are difficult to distinguish from the surrounding structures . Tasneem et al.11) described a case involving a radiolucent intracranial wooden foreign body and the usefulness of the lung window setting for detecting wooden foreign bodies on ct images . Careful review of ct images with the lung window setting can be helpful for detecting a wooden foreign body . Unlike ct images, mri is more useful for detecting wooden foreign bodies that appear hypointense on t1- and t2-weighted mri scans and that can be distinguished from air or fat.69) in addition, infected lesions exhibit gadolinium enhancement . The initial ct image of the patient showed a single area of air in the frontal lobe . Steudel and hacker10) reviewed 508 cases with acute head injuries and found that 49 (9.7%) had a pneumocephalus on brain ct scans, with 5 (1%) showing a single air bubble and 6 (1.1%) having an intracerebral or intraventricular location . However, they did not experience a case involving a single air bubble with an intracerebral or intraventricular location . These findings suggest that patients with a pneumocephalus with a rare presentation need to be examined more carefully . Patients with retained intracranial wooden foreign bodies frequently develop delayed infectious complications, and surgical removal is necessary, even in the absence of symptoms.178) our patient experienced late - onset seizures, which could have been secondary to gradual gliosis, progressive granulomatous changes, or delayed abscess formation, as has been noted in cases with retained foreign bodies.2) therefore, if intracranial injury from a wooden foreign body is suspected, careful history taking and imaging are very important . Once the diagnosis of an intracranial foreign body is confirmed, surgical removal of the foreign body is required . The present case illustrates the necessity for special attention to patients suspected of having pneumocephalus with a rare presentation during the initial examination . Early surgical removal of the intracranial foreign body is necessary to prevent complications.
Honey whose medicinal uses date from ancient times has been lately rediscovered as therapy for wounds . The antimicrobial effect of honey has been reported by a number of workers it is commonly used as a base for ointments and has very successfully been applied in surgical dressings for open wounds and burns to avoid septic infections . The current prevalence of antibiotic - resistant microbial species has led to a re - evaluation of the therapeutic use of ancient remedies, including honey . Strong solution of honey or sugar and sugar pastes inhibits microbial growth due to high osmolarity but when used as dressings this action ceases . But it has been traditionally used in treatment of burn wound infection in rural india and the study has shown that it is superior to silver sulfadiazine . Indiscriminate use of antibiotics has led to the emergence of drug - resistant strains which have a significant impact on patient's morbidity and mortality . To date, there are many publications on studies performed both in vitro and in vivo on the therapeutic properties of manuka honey, and have confirmed its activity against a wide range of medically important bacteria . Honey is produced from many floral sources and its antimicrobial activity varies markedly with its origin and processing. [911] this variation can be due to difference in the enzymatic action and in the presence of additional antibacterial components derived from the floral source . As the potential role for honey as a topical agent to manage surgical site or wound infections is increasingly acknowledged other honeys need to be assessed and evaluated . The present study was to evaluate the antimicrobial activity of honey native to india at various concentrations against pseudomonas aeruginosa causing wound infection and its comparison with antibiotics and dettol . A total of 50 strains of p. aeruginosa isolated from different types of wound infection including burns wound were used in the study . The antibiotic - sensitivity patterns of the isolates were studied by kirby bauer's disc diffusion method using commercially available antibiotic discs (hi - media labs, mumbai india) gentamicin (10 g), amikacin (30 g), ceftazidime (30 g), ciprofloxacin (5 g), netillin (30 g), cefotaxime (30 g), piperacillin (100 g), and imipenem (10 g) as per the clinical and laboratory standards institute (clsi) standards . Agmark grade honey (dabur india, capital overseas), a polyfloral honey with yellowish brown color, was used in the study . It had 100% purity and each 100 g of honey contains 80 g natural sugars, sodium 17 mg, potassium 138 mg, calcium 13 mg, iron 1.5 mg, and phosphorus 5 mg; sterilized by ultrafiltration and have the floral source from himalayas, nilgiris, and sunderbans of india . The minimum active dilution of honey against p. aeruginosa isolates was determined by agar dilution methods . Double strength nutrient agar was used for the study, measured out into 10 ml aliquots and autoclaved . To prepare the plates it was melted and maintained at a temperature of 50 c water - bath until poured . Solutions of the honey samples (at a concentration of 50% v / v) were prepared in sterile de - ionized water immediately prior to performing an assay . Appropriate concentrations of the stock and deionized water (total 10 ml) were dispensed aseptically into 10 ml sterile double strength nutrient agar before pouring into petri dishes to produce a dilution series (5 - 25%) from concentrations required in a volume of 20 ml . Antibacterial action of honey was compared with dettol, (reckitt benckiser india ltd, capital overseas) at a concentration similar to honey which contains an aromatic chemical compound chloroxylenol (c8h9clo) (4.8% of the total dettol mixture), which is the key ingredient exhibiting unique antiseptic property . The turbidity was adjusted to 0.5 mc farland and 10 l of culture was spot inoculated on to the surface of the medium . The plates were incubated at 37c for 24 h before visual assessment of the growth . Control plates of plain nutrient agar were included in each susceptibility assay to confirm the viability and density of the cultures . Aeruginosa american type culture collection (atcc) strain 27853 was inoculated on each plate for comparison . Honey was used undiluted and at different dilutions . With each experiment saline control of the organism two milliliters each of undiluted and different dilutions of the honey were taken in sterile test tubes and inoculated with 20 l of broth culture of the test bacterium in an initial concentration of approximately 510 cfu / ml . All the tubes were incubated at 37c and the viable count of bacteria in each tube was determined at an interval of 4 h up to 24 h by the surface plate method . Viable count of bacteria in both test and control tubes at each time interval was noted by performing plate culture on nutrient agar without honey . Agmark grade honey (dabur india, capital overseas), a polyfloral honey with yellowish brown color, was used in the study . It had 100% purity and each 100 g of honey contains 80 g natural sugars, sodium 17 mg, potassium 138 mg, calcium 13 mg, iron 1.5 mg, and phosphorus 5 mg; sterilized by ultrafiltration and have the floral source from himalayas, nilgiris, and sunderbans of india . The minimum active dilution of honey against p. aeruginosa isolates was determined by agar dilution methods . Double strength nutrient agar was used for the study, measured out into 10 ml aliquots and autoclaved . To prepare the plates it was melted and maintained at a temperature of 50 c water - bath until poured . Solutions of the honey samples (at a concentration of 50% v / v) were prepared in sterile de - ionized water immediately prior to performing an assay . Appropriate concentrations of the stock and deionized water (total 10 ml) were dispensed aseptically into 10 ml sterile double strength nutrient agar before pouring into petri dishes to produce a dilution series (5 - 25%) from concentrations required in a volume of 20 ml . Antibacterial action of honey was compared with dettol, (reckitt benckiser india ltd, capital overseas) at a concentration similar to honey which contains an aromatic chemical compound chloroxylenol (c8h9clo) (4.8% of the total dettol mixture), which is the key ingredient exhibiting unique antiseptic property . The turbidity was adjusted to 0.5 mc farland and 10 l of culture was spot inoculated on to the surface of the medium . The plates were incubated at 37c for 24 h before visual assessment of the growth . Control plates of plain nutrient agar were included in each susceptibility assay to confirm the viability and density of the cultures . Aeruginosa american type culture collection (atcc) strain 27853 was inoculated on each plate for comparison . Honey was used undiluted and at different dilutions . With each experiment saline control of the organism two milliliters each of undiluted and different dilutions of the honey were taken in sterile test tubes and inoculated with 20 l of broth culture of the test bacterium in an initial concentration of approximately 510 cfu / ml . All the tubes were incubated at 37c and the viable count of bacteria in each tube was determined at an interval of 4 h up to 24 h by the surface plate method . Viable count of bacteria in both test and control tubes at each time interval was noted by performing plate culture on nutrient agar without honey . A total of 50 strains of p. aeruginosa isolated from infected wounds were studied over a period of 2 years . The antibiotic susceptibility patterns of the p. aeruginosa isolates are shown in figure 1, where the y axis indicates the percentage of the isolates susceptible to various antibiotics plotted on the x axis . Multidrug resistance was a common feature among most of these isolates 41 (82%) and the remaining 9 (18%) isolates were pan sensitive . Among the antibiotics tested against p. aeruginosa isolates in this study imipenem 50 (100%) being highly effective followed by piperacillin 39 (78%), amikacin 38 (76%) netillin 32 (64%), ceftazidime 28 (56%), gentamicin 23 (46%), cefotaxime 23 (46%), and ciprofloxacin 20 (40%), respectively . Percentage of susceptibility pattern to different class of antibiotics to pseudomonas aeruginosa isolates used in the study the effects of honey on all the isolates of p. aeruginosa were studied by the determination of minimum inhibitory concentration (mic), indicating the highest dilution of honey in the culture medium which inhibits the growth of p. aeruginosa isolates . The mic of all the 50 strains (100%) to honey was found to be 20%, of which 19 strains (38%) were having an mic of 15% including the standard strain of p. aeruginosa atcc 27853 . Honey concentrations ranging from 5% to 10% were found to be ineffective as shown in figure 2 . The y axis indicates the number susceptible percentage of isolates to honey and dettol at various concentrations (5 - 25%) plotted on the x axis . Mic of pseudomonas aeruginosa isolates towards honey and dettol at different concentrations the time kill assay for the bactericidal effect was tested on five isolates of p. aeruginosa so as to understand the time required to kill the bacterial population irrespective of their antibiotic susceptibility patterns as isolates varying in their susceptibility pattern were used . Honey at concentrations of 20%, 25%, 50% could bring out complete destruction in 24 h, whereas concentration of honey at 75% and 100% could bring about complete destruction of p. aeruginosa in 12 h [table 1]. The lowest concentration of the honey which showed bactericidal activity was at 20% for all the five isolates tested in time kill assay . The antibacterial activity of honey was concentration dependent for all the 50 isolates tested at a concentration below 20% using agar dilution methods [figure 2]. The antibacterial effect of 50% honey on p. aeruginosa with number of hours and percentage of survival rate was calculated . The average percentages of survival rate for five p. aeruginosa isolates after 4 h, 8 h, 12 h, and 24 h were found to be 22.3, 5.2, 1.1, and 0 respectively as shown in table 2 . Time kill assay of honey against the wound isolates of pseudomonas aeruginosa antibacterial effect of 50% honey on p. aeruginosa wound isolates the present study shows the bactericidal activity of honey against p. aeruginosa strains . As honey is considered as a natural antiseptic in the management of wound infections, its efficacy was compared with dettol . It was observed that these organisms were more or less susceptible to honey and dettol at 20% and 10% irrespective to their antibiotic sensitivity patterns . Dettol antiseptic liquid contains chloroxylenol which is proved to kill a wide variety of microbes and is widely used for cleansing wound . The antibacterial action is due to disruption of the cell membrane potentials and blocking the production of adenosine triphosphate . The antibacterial effect of honey was concentration dependent and bactericidal effect was observed at concentration of 20% or more for p. aeruginosa isolates tested . Since p. aeruginosa are recalcitrant to antibiotic therapy the ability to inhibit test isolates irrespective of their antibiotic sensitivity patterns has important clinical applications . This property may make honey useful in the treatment of drug - resistant infections . In this study, we found that indian origin honey have activity against p. aeruginosa wound isolates comparable to tualang and manuka honey as reported in previous studies . Reported that any honey can have equivalent antibacterial activity against some bacteria compared with other pharmaceutical honeys, whereas basson et al . Found no such high antimicrobial activity for honeys native to south africa . Compared the activity of ulmo90 and manuka honey and reported similar activity against the p. aeruginosa at a concentration of 12.5%, better than the results from our study . Results from these studies confirm that honeys from different countries and regions may have wide variations in their antimicrobial activity . It has been shown that honey may have antimicrobial action ranging from lesser than 3% to 50% and higher concentrations . Honey has been successfully used in the treatment of surgical wounds, burns wound, and decubitus ulcers . It has also been shown as a good medium to store skin grafts and honey has antileishmanial effect also . High sugar concentration, low ph, hydrogen peroxide generation, proteinaceous compounds, or other unidentified components present in the honey may all provide antimicrobial activity . Shrinkage and disruption of the bacteria may be due to its osmotic effect, low ph, and also due to the presence of antibacterial substance such as inhibine . Several components may contribute to the non - peroxide activities of honey, such as the presence of methyl syringate and methylglyoxal, which have been extensively studied in leptospermum honeys . Besides its antimicrobial properties, honey can clear infection in a number of ways in vivo, like boosting the immune system, anti - inflammatory, and antioxidant activities and via stimulation of cell growth . After reviewing the literature it has been found that apart from mulai et al ., no other indian study has been carried out exclusively on antibacterial activity of honey against p. aeruginosa in vitro . As there is lack of scientific research and documentation, still the medicinal properties of indian honeys further studies on human subjects are required in vivo to understand the efficacy of indian honeys in eliminating bacteria from wounds . As this study presents the findings of in vitro antibacterial activity of honey against planktonic bacteria this should not be related to chronic wound environment where indeed biofilms of p. aeruginosa may be present and the characteristic of bacteria can change; hence, future studies in this direction will pave the way in establishing the medicinal importance of indian honey against the sessile forms of p. aeruginosa . As honey is easily available in the market and is inexpensive its antibacterial activity was comparable to other medicinal honeys . All strains of p. aeruginosa including both resistant phenotypes and sensitive strains were inhibited at 20% antibacterial honey concentrations in vitro . This intriguing observation may have important clinical implications and could lead to a new approach for treating multidrug resistant p. aeruginosa infected wounds using honey of indian origin.
Metastatic spread of tumor cells detached from melanoma into the central nervous system (cns) occurs haematogenically since lymphatic drainage is absent in the brain . The blood - brain barrier is usually intact in metastases that are smaller than 0.25 mm in diameter . The cells from brain metastases show a slower growth rate and exhibit lower metastatic potential than cells from visceral metastases, indicating that brain metastases do not necessarily represent the end stage in the metastatical cascade . Rather, brain metastases are likely to originate from a unique subpopulation of cells within the primary neoplasm . Some authors found an association between the size of the cerebral metastatic lesion from malignant melanoma and clinical parameters characteristic of tumor behavior . They classified the metastases from melanomas to their size: smaller than 1 cm (group a), between 1 and 4 cm (group b), and bigger than 4 cm (group c), in order to assess the clinical course of the disease and predict the response to treatment . Group b lesions are the most common, independent of the site of the primary tumor, except for patients with rectal melanoma . Asymptomatic patients usually have group a metastases, whereas those with nonspecific complaints or behavioural changes usually have group b metastases . Solitary lesions usually belong to groups b or c, whereas multiple lesions belong mainly to groups a or b . Cns metastases occur in 10 to 40% of melanoma patients in clinical studies and up to 90% in autopsy studies . In 15% to 20% of these patients, a second organ is involved, and in 20% three organs are involved [47]. The cumulative risk at 5 years for patients with melanoma to develop cns metastases corresponds to about 7% . Fifty - eight percent of the patients with intracranial metastasis in malignant melanoma are male and 42% were female . Malignant melanoma represents the third most common cause for central nervous system metastases after breast and lung cancer . Whereas breast, lung, and kidney metastases are predominantly solitaire, malignant melanoma metastasizes often in a multiple way nevertheless, only about 5% of the patients with multiple melanoma metastases have more than five intracerebral metastatical lesions . Seventy - one percent of the primary lesions are invasive lesions with mean greater than thickness of 3.5 mm . Nevertheless, the studies have showed a significative prevalence of small and well - circumscribed lesions at surgical aspect . The case of disseminated carcinomatous cell spreading throughout the brain is called military metastases or carcinomatous encephalitis . Most patients with advanced military metastases will have widespread extracranial disease, but the majority will die from intracerebral spread . Risk factors for central nervous system (cns) metastases among patients with coetaneous malignant melanoma are: male, head and neck or oral primary lesion, presence of visceral metastases, mainly lung, primary tumor thickness and ulceration of primary lesion . One of the many features of the malignant melanoma phenotype, in vitro and in vivo, is the elevated heparanase production and activity, which confers the capacity of degrading the subendothelial matrix produced by endothelial cell monolayer cultures . Supra - additive levels of heparanase activity are found when brain endothelial cells are coincubated with brain - metastatical melanoma cells in equicellular amounts . Murine and human brain - metastatical melanoma cells solubilize sulfated matrix proteoglycans at levels significantly higher than their parental lines . Sulfated matrix proteoglycans are rich in heparan sulfate (hspgs), with minor amounts of chondroitin and dermatan sulfates . The pattern of hspg degradation by brain - metastatical melanoma cells differs from that of less metastatical parental cells or cells metastatical to organs other than the brain . Cooperative interactions between heparanases from tumor and endothelial sources are suggested to be a significative mechanism in the invasion process . The brain is a unique microenvironment enclosed by the skull and maintaining a highly regulated vascular transport barrier . To metastasize to the brain, malignant tumor cells must attach to microvessel endothelial cells, invade the blood - brain barrier (bbb), and respond to brain survival and growth factors . Neurotrophins (nts) are important in brain invasion because they stimulate this process . In brain - metastatic melanoma cells, nts can promote invasion by enhancing the production of extracellular matrix degradative enzymes such as heparanase, an enzyme capable of locally destroying both the extracellular matrix and the basement membrane of the bbb . Melanoma cell lines exhibiting low ability to form brain metastases express low - affinity neurotrophin receptor p75ntr in relation to their brain - metastatic potentials . Presence of functional trkc, the putative receptor for the invasion - promoting neurotrophin nt-3, is also expressed in brain - metastatic potential cells . Brain - metastatic melanoma cells can also produce autocrine factors and inhibitors that influence their growth, invasion, and survival in the brain . Synthesis of these factors may influence nts production by brain cells adjacent to the neoplastic invasion front, such as oligodendrocytes and astrocytes . In brain biopsies, increased amounts of nerve growth factor (ngf) and nt-3 were observed in tumor - adjacent tissues at the invasion front of human melanoma tumors . Astrocytes are supposed to contribute to the brain - metastatic specifity of melanoma cells by producing nts - regulated heparanase . Trophic, autocrine, and paracrine growth factors may, therefore, determine whether metastatical cells can successfully invade, colonize, and grow in the central nervous system . Integrin alpha(v)beta(3) is a molecule of adhesion to the endothelium, and its expression on the metastatical pattern of human melanoma cells in the central nervous system (cns) is already studied . Although it is predicted that the adhesion of tumor cells to endothelial cells plays a role in this phenomenon, tumor cell alpha(v)beta(3) integrand expression per se does not explain the difference in metastatic behavior in the cns . Headache is the most common presenting symptom, but brain metastases should be suspected in all melanoma patients with new neurological findings . In patients with brain metastasis, melanoma is one of the primaries cancers with the highest frequency of seizures, found in about one third of the patients . Mean time interval between the initial diagnosis of melanoma and development of first symptoms of cns metastases is 3.5 years . The time from diagnosis of the primary tumor to discovery of disease in the cns is significantly longer for those who had group a lesions (metastases smaller than 1 cm), compared with those who had groups b (metastases between 1 and 1.4 cm) or c lesions (metastases bigger than 4 cm) [17, 18]. In patients whose disease had progressed to brain metastases, freedom from such metastases decreases logarithmically with time from initial presentation . This suggests a random distribution of progression rates with a mean time of 2.5 years between diagnosis and development of intracranial metastases . All patient with new neurological signs and a previous primary melanoma lesion must be investigated . Ct scan finds solitary lesions in 54.2% and multiple lesions in 45.8% of patients with malignant melanoma cerebral metastasis . Eighty - four percent of the solitary lesions are located in the cerebral hemispheres with 62.5% of these in the frontal region . Seventy - five percent of the cns metastatic melanoma lesions appear on noncontrast study as increased density; 22% are hypodense, and 3% are isodense . All lesions show contrast enhancement, usually appearing as a homogeneous nodular or ring pattern . Mri is the best diagnostic technique for detecting cns metastases (figure 1). However, large, solitary, necrotic metastases can be indistinguishable from high - grade astrocytomas . Using conventional mri, the demonstration of an elevated rcbv (relative cerebral blood volume cbv the ratio between the normal adjacent and the pathological area) may suggest a hypervascular lesion such as renal carcinoma or melanoma . Most commonly, cns melanoma metastasis appears in mri as hyperintense (figure 2) on t1-weighted images and hypointense on t2-weighted images . Hemorrhage in the lesion may have a greater influence on this unique appearance than does melanin . The increased tissue sensitivity of mri allow for 22% of patients greater lesion detection than did ct . Intraoperative identification of brain tumors and tumor margins has been limited by either the resolution of the in vivo imaging technique or the time required to obtain histological specimens . Optical coherence tomography (oct) is a new, noncontact, high - speed and high - resolution, real - time, intraoperative imaging technique, capable of resolutions on micrometer scale, which has been used to identify intracortical melanomas . Oct is analogous to ultrasound b - mode imaging, except that reflections of infrared light, rather than sound, are detected . Oct uses inherent tissue contrast, rather than enhancement with dyes, to differentiate tissue types . The compact, fiber - optic - based design is readily integrated with surgical instruments . Two - dimensional images show increased optical backscatter from regions of metastatic melanoma tumors, which are quantitatively used to determine the tumor margin . Three - dimensional reconstructions reveal regions of tumor penetrating normal cortex and can be re - cut at arbitrary planes . The literature reports suggest that oct can effectively differentiate normal cortex from intracortical melanoma based on variations in optical backscatter . High - resolution and high - speed imaging capabilities of oct may permit the intraoperative identification of tumor and more precise localization of tumor margins . Detection and diagnosis of human malignant melanoma by positron emission tomography (pet) using 18f-10b - l - bpa, a specific melanogenesis - seeking compound, has been developed . This resulted in a novel, highly effective methodology for the selective three dimensional imaging of metastatic malignant melanomas, and for accurate determination of 18f-10b concentration in the tumor and surrounding tissue, providing almost all diagnostic information necessary for complete noninvasive radiation dose planning in the treatment of malignant melanoma . The literature data show that iofetamine (i 123) cintilography can be a good method to differential diagnostic in patients with risk for metastatic melanoma lesions . In patients with metastatic melanoma lesions studies suggest that certain brain tumors such as melanoma are capable of selectively binding iofetamine i 123 because of specific chemical properties of this radiopharmaceutical compound . Intermediate filament keratin is regarded as a good marker for epithelial and mesothelial tumors . In the intracranial and intraspinal spaces keratin has been demonstrated only in the endocrine cells of the adenohypophysis, squamous epithelial islands in the pars tuberalis of the hypophysis, and in the choroid plexus epithelium . Since gliomas and meningiomas do not express keratin, immunohistochemistry essay for keratin provide an additional help for differentiating between metastatic melanomas and primary central nervous system tumors . The addition of monoclonal antibody immunocytology to conventional techniques significantly improves the sensitivity of csf cytology . This is particularly useful in the diagnosis of neoplastic meningitis, also called metastatic meningeal melanomatosis (mmm) a complication of malignant melanoma disease whose diagnosis rests on the demonstration of malignant cells within the csf . The putative csf tumor markers, fibronectin, and beta 2-microglobulin, are elevated significantly in mmm (metastatic meningeal melanomatosis) but not in patients with solid cerebral metastases . A prominent increase in the igm index, which reflects intrathecal b - cell stimulation, and rise of igg index, interleukin-6, and tumor necrosis factor - alpha in mmm patients provide preliminary evidence for a local intrathecal immune response triggered by melanoma cell invasion of the subarachnoid space . Thus, more sensitive diagnostic approaches are needed to identify subclinical cns metastases . Currently, standard cytological analysis of the cerebrospinal fluid (csf) is limited by its poor sensitivity . A more sensitive assay was therefore developed using multiple reverse transcriptase - polymerase chain reaction (rt - pcr) markers . The csf is collected and assessed by rt - pcr for three known melanoma - associated markers (mage-3, mart-1, and tyrosinase) to detect occult metastatic melanoma cells in the csf . The correlation between csf rt - pcr positivity of mart-1 and/or mage-3 and the development of cns metastases is significant . Of the patients with positive csf rt - pcr markers, often surgery, radiosurgery, whole brain radiotherapy and chemotherapy are used in combination to obtain longer remissions and optimal symptom relieve . Patients with limited cns metastases and widespread systemic disease can achieve prolonged survival with targeted treatment of cns lesions and aggressive systemic therapy chemotherapy . Gamma knife radiosurgery or surgical resection of cns disease prior to chemotherapy improves survival versus delayed treatment in patients melanoma with melanoma brain metastases . Radiotherapy is recommended to those cases where total resection were not possible and the concomitant use of corticoid pulse therapy is recommended in order to reduce peritumoral edema and mass tumor effect . Surgery of an isolated metastasis can lead to a long survival but brain lesions are frequently numerous and associated with an extracerebral diffusion (figure 3). Complete surgical resection of intracranial melanoma metastatic lesions results in a mean survival period of 10.3 months . Patients with primary lesions of the head and neck have lowest mean survival, about 3.3 months, whereas those whose primary sites are unknown have the longest mean survival, approximately 7.5 months . Duration and quality of survival depend on the extent of metastatic disease and response to treatment . Treatment goals with wbi are palliation of symptoms and prolongation of life . Although brain metastases may be treated with surgery and/or stereotactic radiosurgery (srs) when disease is limited to three lesions, treatment for patients with large or multiple metastases is limited to wbi . While formal response and survival analyses of the impact of wbi in melanoma metastases have not been reported, the estimated mean survival time for unselected patients with cns metastases is only 2 to 4 months, with 1-year survival rates of less than 13% . This rates prevent the use of wbi as single therapeutic option . In a selected population of patients with limited cns involvement, overall survival is significantly improved in patients with multiple metastasis who receive adjunctive cranial irradiation versus those who had surgery alone . Therefore, adjunctive cranial irradiation is justified for melanoma patients who undergo surgical therapy for solitary brain metastases . Survival in patients presenting with solitary brain metastases is improved by a reduction of relapse in the brain as a component of failure by combined surgery and irradiation . In these patients survival will depend basically upon control of systemic disease some authors even suggest the prophylactic whole brain irradiation for patients with melanoma that present high risk of metastases, once cns metastases are sometimes the sole site of clinical relapse, and are frequently disabling . In accelerated irradiation regimens, the total tumor dose varies from 3000 to 4800 rad, and the overall treatment time from 1 to 2 weeks . This more aggressive form of treatment has demonstrated no significant improvement in the results from accelerated fractionation in the treatment of intracranial metastases . The result of the radiotherapy treatment did not depend on the site of the primary lesion, number of intracranial metastases, total dose, or the dose perfraction . There are, however, two subgroups not mutually exclusive, that benefit significantly from the accelerated fractionation: patients who had a complete resection of brain metastases, and those having no detectable extracranial metastases at the time of their treatment for intracranial metastases . Although in larger metastases the blood - brain barrier is leaky, lesions are resistant to many chemotherapeutic drugs . While systemic therapy for metastatic melanoma produces objective responses in 15% to 50% of patients, available drugs do not penetrate well into the cns, and these patients rarely benefit from systemic therapy . (a) dacarbazinedacarbazine (dtic) gives a mean response rate of 21% on visceral localizations but does not cross the blood brain barrier (bbb). Biological response modifiers like interleukin 2 (il2) leads to a 25% response rate in disseminated melanoma . Dacarbazine (dtic) gives a mean response rate of 21% on visceral localizations but does not cross the blood brain barrier (bbb). Biological response modifiers like interleukin 2 (il2) leads to a 25% response rate in disseminated melanoma . (b) pcnuthe results indicate that systemic pcnu is unlikely to be more effective than other currently used chemotherapy in patients with malignant melanoma and cns metastases . Moreover, treatment with pcnu presents frequently some acute complications like granulocytopenia or thrombocytopenia . The results indicate that systemic pcnu is unlikely to be more effective than other currently used chemotherapy in patients with malignant melanoma and cns metastases . Moreover, treatment with pcnu presents frequently some acute complications like granulocytopenia or thrombocytopenia . (c) temozolomidetemozolomide (tmz) is a novel oral alkylating similar to dacarbazine (the most active single agent in primary melanoma) that have 100% oral bioavailability and considerable penetration of cns tissue . Tmz has broad preclinical antitumoral activity that in melanoma is comparable to that of dacarbazine.sites of remission of metastatic melanomas treated with temozolomide include brain, lung, liver, lymph nodes and muscle . However some few patients may present complications due to severe leucopoenia and thrombocytopenia (who grade 3 and 4). Thus, termozolomide represents a safe treatment option in patients with metastatic melanoma and poor prognosis . Temozolomide (tmz) is a novel oral alkylating similar to dacarbazine (the most active single agent in primary melanoma) that have 100% oral bioavailability and considerable penetration of cns tissue . Tmz has broad preclinical antitumoral activity that in melanoma is comparable to that of dacarbazine.sites of remission of metastatic melanomas treated with temozolomide include brain, lung, liver, lymph nodes and muscle . However some few patients may present complications due to severe leucopoenia and thrombocytopenia (who grade 3 and 4). Thus, termozolomide represents a safe treatment option in patients with metastatic melanoma and poor prognosis . (d) temozolomide combinationstemozolomide (tmz) may also be used in combination with interferon alpha-2b (ifn - alpha2b). The most common adverse events during the use of this association are fatigue, fever, nausea / emesis, anxiety, and diarrhea . The maximum tolerated dose is either tmz 150 mg / m(2) plus ifn - alpha2b 7.5 miu / m(2) or tmz 200 mg / m(2) plus ifn - alpha2b 5.0 miu / m(2). The pharmacokinetics of tmz is not affected by coadministration of ifn - alpha2b [33, 34]. The combination of tmz and whole brain irradiation (wbi) was studied in patients with cns metastatic malignant melanoma, and it was found that, although tmz can be safely administered with wbi, the combination has limited antitumor activity . Another option for the chemotherapy in patients with metastatic melanoma is the use of termozolomide (tmz) p.o . Low - dose interleukin-2 (il2), granulocyte - monocyte colony stimulating factor (gm - csf), and interferon - alpha 2b (ifn alpha). The main toxicity of this combined chemotherapy is the flu - like syndrome and transient liver function disturbances . The mean overall survival with temozolomide reported is 7 months since beginning of therapy . Temozolomide (tmz) may also be used in combination with interferon alpha-2b (ifn - alpha2b). The most common adverse events during the use of this association are fatigue, fever, nausea / emesis, anxiety, and diarrhea . The maximum tolerated dose is either tmz 150 mg / m(2) plus ifn - alpha2b 7.5 miu / m(2) or tmz 200 mg / m(2) plus ifn - alpha2b 5.0 miu / m(2). The pharmacokinetics of tmz is not affected by coadministration of ifn - alpha2b [33, 34]. The combination of tmz and whole brain irradiation (wbi) was studied in patients with cns metastatic malignant melanoma, and it was found that, although tmz can be safely administered with wbi, the combination has limited antitumor activity . Another option for the chemotherapy in patients with metastatic melanoma is the use of termozolomide (tmz) p.o . Low - dose interleukin-2 (il2), granulocyte - monocyte colony stimulating factor (gm - csf), and interferon - alpha 2b (ifn alpha). The main toxicity of this combined chemotherapy is the flu - like syndrome and transient liver function disturbances . The mean overall survival with temozolomide reported is 7 months since beginning of therapy . (e) fotemustine (muphoran)fotemustine (muphoran), a new amino acid - linked nitrosourea, can give a response rate up to 28.2% in patients with cerebral metastases, and the increased survival of responding patients is significant [6, 29]. All responders to fotemustine have mainly cortical, group a (lesion smaller than 1 cm), or group b lesions (lesion s between 1.1 and 4 cm). Patients with group c metastasis (lesion bigger than 4 cm) or leptomeningeal spread do not respond to fotemustine . Fotemustine (muphoran), a new amino acid - linked nitrosourea, can give a response rate up to 28.2% in patients with cerebral metastases, and the increased survival of responding patients is significant [6, 29]. Have mainly cortical, group a (lesion smaller than 1 cm), or group b lesions (lesion s between 1.1 and 4 cm). Patients with group c metastasis (lesion bigger than 4 cm) or leptomeningeal spread do not respond to fotemustine . Cisplatin - based chemotherapy may be useful for palliation in selected patients with malignant melanoma and cns metastases . Intracarotid cisplatin 4075mg / m can be administered alone, with 1,3-bis(2-chloroethyl)-1-nitrosourea (bcnu) or with bleomycin . Thirty percent of the patients have objective improvement in ct scans and about 13% of them have stabilization of disease . Intracarotid cisplatin - based chemotherapy may be useful for palliation in selected patients with malignant melanoma and cns metastases . Intracarotid cisplatin 4075mg / m can be administered alone, with 1,3-bis(2-chloroethyl)-1-nitrosourea (bcnu) or with bleomycin . Thirty percent of the patients have objective improvement in ct scans and about 13% of them have stabilization of disease . (g) interferoninterferon apparently is inactive against melanoma brain metastases, but does cause cns symptoms . More recently, stereotactic radiosurgery (srs) has been used as an effective local treatment for patients with cns melanoma metastases . In several retrospective reports, treatment with srs alone or in combination with whole brain irradiation (wbi) has demonstrated to prolong mean survival . As happens with the treatment with wbi alone, most of the patients die from progressive extracranial disease with locally controlled cns disease [6, 40]. Gamma knife surgery (gks) is effective in treating melanoma metastases in the brain . It appears that the radiobiology of a single high dose overcomes the radioresistance barrier, yielding better results than fractionated radiation . Twenty - four percent of the lesions treated with gks disappear, 35% shrink, 23% remain unchanged, and 18% increasing size . The mean survival time calculated is 10.4 months from the time of treatment with gks . Solitary brain lesions and lack of visceral metastases are statistically predictive of a better prognosis . Boron neutron capture therapy (bnct) is yet an incipient therapy method that has shown good results in rats experiments, mainly if combined by either cereport (rmp-7) mediated modulation of blood - brain barrier (bbb) permeability or hyperosmotic mannitol - induced bbb disruption using borono - phenylalanine (bpa) as the capture agent . Optimizing the delivery of bpa by means of intracarotid injection combined with opening the bbb by infusing cereport or a hyperosmotic solution of mannitol significantly enhance survival times and produce long - term cures in animals with cns melanoma metastasis . These observations are relevant to future clinical studies using bnct for the treatment of intracerebral melanoma . Prognosis for patients with melanoma brain metastasis is still poor with a mean survival time of 6 months after diagnosis [1, 2, 5, 42]. Mean survival in patients treated with chemotherapy is approximately 8.3 months1 year survival of 41% . Estimated mean survival time for patients with cns melanoma metastases treated with whole brain irradiation is only 2 to 4 months . Most of the symptoms of cns melanoma metastasis are unspecific and depend on localization of the lesion . Mri must be used to study more precisely the characteristics of the lesions in order to embase the surgical plane . It is important that the clinician suspect of the possibility of cns metastasis in all patients with new neurological signs and previous primary melanoma lesion . These patients deserve a careful clinical analysis and further exams for investigation (table 1). Often surgery, radiosurgery, whole brain radiotherapy and chemotherapy are used in combination to obtain longer remissions and optimal symptom relieve (table 2). Patients with limited cns metastases and widespread systemic disease can achieve prolonged survival with targeted treatment of cns lesions and aggressive systemic chemotherapy, when clinical conditions of patient allow . Stereotactic radiosurgery is recommended to those cases where total resection are not possible and, in this case, the concomitant use of corticoid pulse therapy may be useful in order to reduce peritumoral edema and mass tumor effect . We highly recommend new brain metastases treatment guidelines issued by the american association of neurological surgeons & the congress of neurological surgeons published in the journal of neuro - oncology in 2009 . It deals with specific issues regarding the level of evidence of chemotherapy, radiosurgery, surgical resection, whole brain radiotherapy, use of corticosteroids, and anticonvulsants in the treatment of brain metastases.
The did is a new tool incorporating different functions; it is a carbohydrate / insulin bolus calculator, an information technology device, and a telemedicine system based on the communication between a health care professional (physician or dietitian) and a patient via text messages . It allows patients to manage a flexible diet and to calculate the matching insulin bolus at each meal . In addition, it includes an algorithm for the calculation of basal insulin dose, based on the values of fasting blood glucose and the presence of hypoglycemic episodes . Did consists of software to be installed in the patient's mobile telephone and enables the phone to be used as a small computer to record the blood glucose values and dose of insulin injections in real time; the system is also able to suggest the daily carbohydrate intake, summing the amount of carbohydrate consumed progressively (fig . 1). Every patient can decide what to eat during the meal, choosing between all the foods listed in the software; the quantification of the total calories and carbohydrate consumed is facilitated by a list of pictures showing the specific food and the amount ingested . The carbohydrate - to - insulin ratio and the glycemic correction factor, identified and prescribed by the health care professional, together with other information already filled out in the did (e.g., physical activity, glycemic target, insulin dose, and specific events), allow it to automatically calculate and suggest the most appropriate insulin dose to be injected . Besides the collection of data on blood glucose measurements, carbohydrate intake, and insulin doses, the use of did is associated with regular feedback for the patient . In fact, data stored in the mobile phone are periodically sent as short text messages and reviewed on the personal computer of the physician . Then, any new therapeutic and behavioral prescription can be sent from the computer to the mobile phone, improving the communication between patients and physician . The did study was an open - label, international, multicenter, randomized (1:1), parallel - group study, having the primary goal of evaluating whether the use of did could improve glycemic control (a1c) in a shorter time and more easily than the carbohydrate counting standard educational approach . Secondary end points were changes in fasting blood glucose (fbg) levels, body weight, lipid profile (serum total cholesterol, hdl cholesterol, ldl cholesterol, and triglycerides), and blood pressure; furthermore, safety - related problems (frequency of hypoglycemic episodes and hospitalizations) and differences in time dedicated to educational activities were taken into consideration . Finally, quality of life and patient treatment satisfaction were investigated in the subgroup of italian patients . The study involved seven diabetes outpatient clinics: three in italy, two in england, and two in spain . Every center was asked to enroll 20 patients satisfying all of the following inclusion criteria: diagnosis of type 1 diabetes, age 18 years, no previous education on carbohydrate counting, and treatment with multiple daily injections of short - acting and long - acting insulin analogs or with continuous subcutaneous insulin infusion; patients practiced self - monitoring of blood glucose at least three times a day . Other important requirements in the selection of patients were adequate familiarity in the use of mobile phones, according to the physician judgment, and possession of a personal mobile phone card . All of the patients were requested to give written informed consent to gain entrance to the study . Patients were excluded if they were being treated with nph insulin or soluble regular insulin, had an eating disorder, were pregnant, were unable to send or receive short text messages, were unable or unwilling to give informed consent, or had any other disease or condition that may interfere with compliance with the protocol or completion of the study . Eligible patients were randomly assigned to start the standard carbohydrate counting educational program or the did approach . Patients randomly assigned to the experimental group attended a course on the use of did lasting up to 2 weeks . The course was provided as an outpatient program of three encounters with the physician and/or dietitian . Patients randomly assigned to the control group received the standard educational approach usually used in the center, lasting up to 3 months . Before the start of the study, an investigators' meeting was organized to establish some fundamental rules in the educational training and in the prescription of carbohydrate - to - insulin ratio and the correction factor . At study entry (visit 0), at 3 months (visit 1), and at 6 months (visit 2) clinical information was collected on case report forms . Baseline information included sociodemographic (age, sex, and highest level of school education reached) and clinical characteristics (diabetes duration, insulin therapy, presence and severity of diabetes complications, comorbidities, and concomitant treatments). Blood pressure, body weight, fbg, a1c, and lipid profile were measured at each visit . Additional information was collected at the end of the study, including the number of contacts between the patient and the diabetes specialist (both short text messages and office visits) and any serious hypoglycemic episode requiring medical intervention . Changes in the health - related quality of life were evaluated in the subgroup of italian patients, using generic (sf-36 health survey [sf-36]) and diabetes - specific (world health organization - diabetes treatment satisfaction questionnaire [who - dtsq]) measures: the sf-36 is one of the most widely used measures of health - related quality of life and consists of 36 items covering eight dimensions: physical functioning, role limitations caused by physical health problems, bodily pain, general health perception, vitality, social functioning, role limitations caused by emotional health problems, and mental health (13). These eight domains may be further aggregated into two summary measures: the physical component summary measure and the mental component summary measure (13). These aggregated scores are transformed to norm - based scores (50 10 mean sd), with higher scores indicating more favorable physical functioning / psychological well - being . The sf-36 has been used in large population studies and in many different clinical conditions, showing excellent psychometric properties (14). It has been translated and validated in several languages, including italian (15).the who - dtsq has been specifically designed to measure satisfaction with diabetes treatment regimens and is appropriate for patients with type 1 and type 2 diabetes (16). The instrument was originally developed to detect changes in satisfaction related to changes in treatment modalities, but it is also appropriate for comparing levels of satisfaction in subjects using different treatment regimens . It is composed of eight items, six of which are summed in a single score ranging from 0 (very dissatisfied) to 36 (very satisfied). The remaining two items are treated individually and explore the perceived frequency of hyperglycemic and hypoglycemic episodes . The who - dtsq has been validated in the italian language among type 1 and type 2 diabetic patients, showing adequate psychometric properties (17). The sf-36 is one of the most widely used measures of health - related quality of life and consists of 36 items covering eight dimensions: physical functioning, role limitations caused by physical health problems, bodily pain, general health perception, vitality, social functioning, role limitations caused by emotional health problems, and mental health (13). These eight domains may be further aggregated into two summary measures: the physical component summary measure and the mental component summary measure (13). These aggregated scores are transformed to norm - based scores (50 10 mean sd), with higher scores indicating more favorable physical functioning / psychological well - being . The sf-36 has been used in large population studies and in many different clinical conditions, showing excellent psychometric properties (14). It has been translated and validated in several languages, including italian (15). The who - dtsq has been specifically designed to measure satisfaction with diabetes treatment regimens and is appropriate for patients with type 1 and type 2 diabetes (16). The instrument was originally developed to detect changes in satisfaction related to changes in treatment modalities, but it is also appropriate for comparing levels of satisfaction in subjects using different treatment regimens . It is composed of eight items, six of which are summed in a single score ranging from 0 (very dissatisfied) to 36 (very satisfied). The remaining two items are treated individually and explore the perceived frequency of hyperglycemic and hypoglycemic episodes . The who - dtsq has been validated in the italian language among type 1 and type 2 diabetic patients, showing adequate psychometric properties (17). Sample size was estimated by assuming a between - group mean difference of 0.5% in a1c levels after 3 months and an a1c sd of 1.0 (as derived from the did pilot study). Given these assumptions, 63 patients per group were needed to ensure a statistical power of 80% (= 0.05). Taking into account a dropout rate of 10%, 70 patients per group had to be enrolled . Analysis was based on all the patients randomized, according to the intention - to - treat principle . For patients lost to follow - up the last information available was used, by means of the last observation carried forward approach . Comparison of a1c and other secondary end points between groups was performed after 3 and 6 months from randomization based on the mann - whitney u test . Within - group differences achieved after 3 and 6 months from randomization were evaluated using the wilcoxon signed - rank test . Because it was hypothesized that the telemedicine approach could help in achieving the desired goals in a shorter period of time, between - groups mean differences at 3 and 6 months the did is a new tool incorporating different functions; it is a carbohydrate / insulin bolus calculator, an information technology device, and a telemedicine system based on the communication between a health care professional (physician or dietitian) and a patient via text messages . It allows patients to manage a flexible diet and to calculate the matching insulin bolus at each meal . In addition, it includes an algorithm for the calculation of basal insulin dose, based on the values of fasting blood glucose and the presence of hypoglycemic episodes . Did consists of software to be installed in the patient's mobile telephone and enables the phone to be used as a small computer to record the blood glucose values and dose of insulin injections in real time; the system is also able to suggest the daily carbohydrate intake, summing the amount of carbohydrate consumed progressively (fig . 1). Every patient can decide what to eat during the meal, choosing between all the foods listed in the software; the quantification of the total calories and carbohydrate consumed is facilitated by a list of pictures showing the specific food and the amount ingested . The carbohydrate - to - insulin ratio and the glycemic correction factor, identified and prescribed by the health care professional, together with other information already filled out in the did (e.g., physical activity, glycemic target, insulin dose, and specific events), allow it to automatically calculate and suggest the most appropriate insulin dose to be injected . Besides the collection of data on blood glucose measurements, carbohydrate intake, and insulin doses, the use of did is associated with regular feedback for the patient . In fact, data stored in the mobile phone are periodically sent as short text messages and reviewed on the personal computer of the physician . Then, any new therapeutic and behavioral prescription can be sent from the computer to the mobile phone, improving the communication between patients and physician . The did study was an open - label, international, multicenter, randomized (1:1), parallel - group study, having the primary goal of evaluating whether the use of did could improve glycemic control (a1c) in a shorter time and more easily than the carbohydrate counting standard educational approach . Secondary end points were changes in fasting blood glucose (fbg) levels, body weight, lipid profile (serum total cholesterol, hdl cholesterol, ldl cholesterol, and triglycerides), and blood pressure; furthermore, safety - related problems (frequency of hypoglycemic episodes and hospitalizations) and differences in time dedicated to educational activities were taken into consideration . Finally, quality of life and patient treatment satisfaction were investigated in the subgroup of italian patients . The study involved seven diabetes outpatient clinics: three in italy, two in england, and two in spain . Every center was asked to enroll 20 patients satisfying all of the following inclusion criteria: diagnosis of type 1 diabetes, age 18 years, no previous education on carbohydrate counting, and treatment with multiple daily injections of short - acting and long - acting insulin analogs or with continuous subcutaneous insulin infusion; patients practiced self - monitoring of blood glucose at least three times a day . Other important requirements in the selection of patients were adequate familiarity in the use of mobile phones, according to the physician judgment, and possession of a personal mobile phone card . All of the patients were requested to give written informed consent to gain entrance to the study . Patients were excluded if they were being treated with nph insulin or soluble regular insulin, had an eating disorder, were pregnant, were unable to send or receive short text messages, were unable or unwilling to give informed consent, or had any other disease or condition that may interfere with compliance with the protocol or completion of the study . Eligible patients were randomly assigned to start the standard carbohydrate counting educational program or the did approach . Random lists were stratified by center . To ensure equal allocation rates within centers, permuted block randomization was used . Patients randomly assigned to the experimental group attended a course on the use of did lasting up to 2 weeks . The course was provided as an outpatient program of three encounters with the physician and/or dietitian . Patients randomly assigned to the control group received the standard educational approach usually used in the center, lasting up to 3 months . Before the start of the study, an investigators' meeting was organized to establish some fundamental rules in the educational training and in the prescription of carbohydrate - to - insulin ratio and the correction factor . At study entry (visit 0), at 3 months (visit 1), and at 6 months (visit 2) clinical information was collected on case report forms . Baseline information included sociodemographic (age, sex, and highest level of school education reached) and clinical characteristics (diabetes duration, insulin therapy, presence and severity of diabetes complications, comorbidities, and concomitant treatments). Blood pressure, body weight, fbg, a1c, and lipid profile were measured at each visit . Additional information was collected at the end of the study, including the number of contacts between the patient and the diabetes specialist (both short text messages and office visits) and any serious hypoglycemic episode requiring medical intervention . Changes in the health - related quality of life were evaluated in the subgroup of italian patients, using generic (sf-36 health survey [sf-36]) and diabetes - specific (world health organization - diabetes treatment satisfaction questionnaire [who - dtsq]) measures: the sf-36 is one of the most widely used measures of health - related quality of life and consists of 36 items covering eight dimensions: physical functioning, role limitations caused by physical health problems, bodily pain, general health perception, vitality, social functioning, role limitations caused by emotional health problems, and mental health (13). These eight domains may be further aggregated into two summary measures: the physical component summary measure and the mental component summary measure (13). These aggregated scores are transformed to norm - based scores (50 10 mean sd), with higher scores indicating more favorable physical functioning / psychological well - being . The sf-36 has been used in large population studies and in many different clinical conditions, showing excellent psychometric properties (14). It has been translated and validated in several languages, including italian (15).the who - dtsq has been specifically designed to measure satisfaction with diabetes treatment regimens and is appropriate for patients with type 1 and type 2 diabetes (16). The instrument was originally developed to detect changes in satisfaction related to changes in treatment modalities, but it is also appropriate for comparing levels of satisfaction in subjects using different treatment regimens . It is composed of eight items, six of which are summed in a single score ranging from 0 (very dissatisfied) to 36 (very satisfied). The remaining two items are treated individually and explore the perceived frequency of hyperglycemic and hypoglycemic episodes . The who - dtsq has been validated in the italian language among type 1 and type 2 diabetic patients, showing adequate psychometric properties (17). The sf-36 is one of the most widely used measures of health - related quality of life and consists of 36 items covering eight dimensions: physical functioning, role limitations caused by physical health problems, bodily pain, general health perception, vitality, social functioning, role limitations caused by emotional health problems, and mental health (13). These eight domains may be further aggregated into two summary measures: the physical component summary measure and the mental component summary measure (13). These aggregated scores are transformed to norm - based scores (50 10 mean sd), with higher scores indicating more favorable physical functioning / psychological well - being . The sf-36 has been used in large population studies and in many different clinical conditions, showing excellent psychometric properties (14). It has been translated and validated in several languages, including italian (15). The who - dtsq has been specifically designed to measure satisfaction with diabetes treatment regimens and is appropriate for patients with type 1 and type 2 diabetes (16). The instrument was originally developed to detect changes in satisfaction related to changes in treatment modalities, but it is also appropriate for comparing levels of satisfaction in subjects using different treatment regimens . It is composed of eight items, six of which are summed in a single score ranging from 0 (very dissatisfied) to 36 (very satisfied). The remaining two items are treated individually and explore the perceived frequency of hyperglycemic and hypoglycemic episodes . The who - dtsq has been validated in the italian language among type 1 and type 2 diabetic patients, showing adequate psychometric properties (17). Sample size was estimated by assuming a between - group mean difference of 0.5% in a1c levels after 3 months and an a1c sd of 1.0 (as derived from the did pilot study). Given these assumptions, 63 patients per group were needed to ensure a statistical power of 80% (= 0.05). Taking into account a dropout rate of 10%, 70 patients per group had to be enrolled . Analysis was based on all the patients randomized, according to the intention - to - treat principle . For patients lost to follow - up the last information available was used, by means of the last observation carried forward approach . Comparison of a1c and other secondary end points between groups was performed after 3 and 6 months from randomization based on the mann - whitney u test . Within - group differences achieved after 3 and 6 months from randomization were evaluated using the wilcoxon signed - rank test . Because it was hypothesized that the telemedicine approach could help in achieving the desired goals in a shorter period of time, between - groups mean differences at 3 and 6 months 1). Fewer patients than those scheduled (130 vs. 140) were involved, as a result of organizational problems in two centers . However, because results show a sd of a1c of 0.76% and the dropout rate was 8.5%, the a posteriori study power to detect a difference between groups of 0.5% in a1c levels was 95% . The study also had a statistical power of 80% to detect a between - groups difference in a1c levels of 0.38% . The two groups did not differ for any sociodemographic and clinical characteristic, with the exception of slightly higher levels of triglycerides in the did group . Patients in the did arm also had a higher prevalence of retinopathy and symptomatic neuropathy, although statistical significance was not reached . Patients' characteristics according to the randomization arm data are means sd or frequency . * p values refer to the test for categorical variables or the mann - whitney u test for continuous variables . Overall, 11 patients dropped out during the study, 2 in the standard group and 9 in the did group (fig . 2 patients found it difficult to use the did system, 4 had difficulties in sending text messages because of poor mobile network coverage in their area, 2 were not compliant with visit scheduling, and 1 moved to another area . Between- and within - group changes after 3 and 6 months are shown in table 2 . Between- and within - group differences in clinical parameters and quality - of - life scores at visit 1 and visit 2 with respect to baseline values data are means sd . 3 months and 6 months columns show the mean variation at visit 1 and visit 2 with respect to baseline (0) values . Questionnaires were administered to a subgroup of 60 patients (30 in the did group and 30 in the standard group). A significant reduction in a1c levels of 0.5% was documented in both groups after 3 months and maintained until the end of study . This improvement in metabolic control was obtained by devoting to carbohydrate counting education a median (range) of 6 (215) h in the did group and 12 (2.525) h in the standard group (p = 0.07). Furthermore, after 6 months there was a nonsignificant decrease in fbg in the did group (from 182.8 85.6 to 162.9 67.0 mg / dl) and a nonsignificant increase in the standard group (from 176.9 68.4 to 186.3 79.1 mg / dl) (between - groups p = 0.13). The increase in body weight was lower in the did group (0.7 3.6 kg) than in the standard group (1.5 2.3 kg), but the difference was not statistically significant (p = 0.22). Furthermore, although we found no differences in mean daily doses of short - acting insulin between the two groups (did group 20.6 8.2 iu / day and standard group 20.1 7.8 iu / day; p = 0.92), mean daily doses of long - acting insulin were lower in the did group than in the standard group, although statistical significance was not reached (did group 17.4 7.4 iu / day and standard group 21.4 10.0 iu / day; p = 0.12). The did group showed a significant decrease in triglyceride levels in comparison with the standard group; no other between - groups changes were documented . The did group generally showed a tendency toward a small, not significant improvement in all the measures considered, whereas in the standard group all parameters, except diastolic blood pressure and hdl cholesterol, tended to slightly increase at the end of the study . No patients in either group were admitted to the hospital during the study, and none reported any severe hypoglycemic episode requiring assistance . In each group, two patients reported episodes of mild hypoglycemia (p = 0.93). The median (range) number of text messages sent by each patient during the study was 52 (675), whereas the number of text messages sent by the physician was 39 (2270). In other words, patients sent about 2 text messages / week to their physician, and the physician regularly replied to confirm the therapeutic scheme or to modify the parameters set in the did (carbohydrate - to - insulin ratio, insulin sensitivity factor, and/or blood glucose target). In terms of costs for the patient, assuming a cost of 1015 cents per message, and considering that on average each patient sent 52 messages, the overall cost sustained did not exceed 8 . Results of quality of life evaluation performed on the subsample of 60 patients enrolled in the italian centers are shown in table 2 . A statistically significant difference in favor of the did group was documented for treatment satisfaction, as expressed by the who - dtsq score . Similarly, the score testing the perceived frequency of hyperglycemic episodes significantly decreased after 3 months in the did group but not in the control group . Several sf-36 subscales (role physical, general health, vitality, and role emotional) also showed significantly higher improvements in the did group than in the standard group . In addition, pre to post within - group comparisons underlined the beneficial effects of did in the experimental group in terms of who - dstq - score, perceived frequency of hyperglycemic episodes, general health perception, and vitality; on the other hand, all scores within the standard group tended to worsen at 3 months, although statistical significance was not reached . The complexity of the educational approach needed to teach carbohydrate counting and consequent insulin adjustment can represent an obstacle for many patients, thus limiting the possibility of its widespread use as an effective self - management tool . The carbohydrate / insulin bolus calculator is coupled with a telemedicine system based on short text messages . At the present time, the most common way of data communication between patient and diabetologist is represented by the paper diary, which is often perceived as a boring document not adequately filled in; furthermore, even if it is sufficiently complete, it cannot facilitate a day - by - day adjustment of the insulin dose and lifestyle (18). In contrast, did is installed on the mobile phone, which is a familiar device already used in daily life by the majority of individuals . Did facilitates not only the automatic storage of blood glucose measurements, carbohydrate intake, and insulin doses but also the exchange of information between patient and care provider via text messages . In this respect, although previous, small studies have evaluated the efficacy of telemedicine systems based mainly on the transmission of self - monitoring of blood glucose values and feedback from the health care provider (10), this is, to our knowledge, the first study investigating a multipurpose instrument, replacing the classic approach to insulin dose modification . Our data show that did can represent a useful device, incorporating several features helping patients promote dietary freedom and flexible insulin bolus . The first pilot study previously showed that the system is safe, easy to use, and well accepted by the vast majority of patients . What these new results add is that the use of did is at least as effective as the traditional educational approach to carbohydrate counting in reducing a1c levels, while producing different concomitant benefits . First, it allowed patients to avoid the complexities of carbohydrate counting and insulin dose adjustment with a halving in the time dedicated to education and thus potentially increasing the proportion of individuals with type 1 diabetes adopting this method . Of note, despite the higher rate of dropouts in the did group, only two patients interrupted the study because of difficulties in using the telecare system, thus confirming that the device can be easily used by the majority of patients . Second, the use of did was associated with lower weight gain, probably because of the requirement of lower doses of long - acting insulin . It is worth mentioning that, despite the use of lower doses of long - acting insulin, patients assigned to the did group showed a reduction in fpg levels during the study, whereas levels slightly increased in the control group . This finding is important in light of the need to adopt therapeutic strategies that achieve good metabolic control while minimizing insulin dosage . Third, the use of did was also associated with a significant improvement in several mental and physical components of the sf-36 health survey, compared with the standard group . This also translated into a marked improvement in treatment satisfaction, thus suggesting that the use of telemedicine can increase the level of acceptance of insulin treatment and help patients cope with the disease first, we were not able to measure the effect of did in reducing glucose variability . In fact, by allowing greater flexibility, one can speculate that telemedicine produced positive effects on postprandial blood glucose excursions also . Second, even if specific guidelines were established in the prestudy investigators' meeting, the did educational intervention was influenced by the individual practice of the different international participating centers, thus varying in duration . Nevertheless, the randomization was stratified by center, making the comparison between telemedicine and usual care unbiased . In summary, did was at least as effective as traditional carbohydrate counting education, allowing dietary freedom to a larger proportion of type 1 diabetic patients . Did larger studies are needed to reach more solid conclusions regarding the effects of did on fbg, body weight, and insulin dosage.
In recent years, obesity caused by the hypertrophy of white adipose tissue (wat) has steadily increased worldwide, and has become a serious social problem . In 2010, the organization for economic cooperation and development (oecd) released a report on the current state of obesity and the cost - effectiveness of preventive measures [2, 3]. That report states that obesity rates have risen in many countries, and that one in two individuals is either obese or overweight in about half of oecd countries . It is widely known that obesity is a risk factor for various lifestyle - related diseases such as type 2 diabetes and hypertension, and that obesity and diabetes cause increases in atherosclerotic disease . Therefore, there is an urgent need to establish strategies for the prevention and improvement of obesity and diabetes . Epidemiological studies have shown that exercise is effective for preventing and improving obesity and diabetes [4, 5]. For example, a study by helmrich et al . Followed 5,990 male graduates of the university of pennsylvania over 14 years and found that the risk of developing diabetes is reduced by 6% for every 500 kcal increase in weekly exercise . Doctors over five years revealed that even a once - weekly bout of exercise at an intensity that is sufficient to cause sweating reduced the risk of developing diabetes . Nurses over eight years showed that the group that exercised at least once a week at an intensity sufficient to cause sweating had a relative risk of developing diabetes of 0.84 compared with a group that exercised less than once a week . Although wat was once considered to be merely a site for energy storage, in recent years it has become better understood at the molecular level; for example, how wat secretes physiologically active substances, collectively known as adipokines, and how obesity - induced dysregulated expression of adipokines in wat causes insulin resistance, which is the pathogenesis of diabetes [911]. Therefore, wat is considered to be one of the tissues that play a critical role in the onset of lifestyle - related diseases, and the reduction of excess wat and the improvement of abnormal adipokine secretion are important strategies for the prevention and improvement of lifestyle - related diseases . Exercise training (tr) not only causes a loss of wat mass, but can also influence the secretory response and expression of adipokines in wat . The major role of subcutaneous and visceral wat is to supply and store energy via adipocytes in wat . Most of the ingested excess energy is stored within adipocytes in the form of triglycerides, which are formed through the binding of glycerol and fatty acids . During exercise, catchecolamines (adrenaline and noradrenaline) secreted from the adrenal medulla or the sympathetic nerve terminal break down triglycerides within adipocytes, and the resultant fatty acids are carried to skeletal muscle via the blood . However, following the discovery of leptin by zhang et al . In 1994, leptin was established as a hormone that is secreted by wat, and a string of new humoral factors that are secreted by wat were discovered . Therefore, the old concept of wat as a mere storage site for energy has been revised to also acknowledge it as an endocrine organ . The humoral factors secreted from wat are collectively referred to as adipokines (figure 1). In recent years, it has become clear that obesity is a chronic and mild systemic inflammatory condition, and there is much evidence that chronic inflammation of wat contributes to the development of insulin resistance . This systemic inflammation has become closely acknowledged as the molecular basis of diabetes [911]. When adipocyte hypertrophy occurs due to excessive energy intake or lack of exercise, infiltration by macrophages, which are one type of immunocompetent cell, the production of proinflammatory adipokines, such as tumor necrosis factor- (tnf-) and monocyte chemoattractant protein 1 (mcp-1), is increased and the production of anti - inflammatory adiponectin is decreased, thereby causing chronic inflammation of wat (figure 2) [1416]. This increase in proinflammatory adipokines is not limited to wat, but also promotes insulin resistance in skeletal muscle and liver as a paracrine agent . Leptin is a hormone that acts on leptin receptors (ob - r) in the hypothalamus to strongly suppress appetite and promote increased energy expenditure [1719]. There is strong ob - r expression in the arcuate nucleus, ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, and lateral hypothalamic area of the hypothalamus . Although the expression of mrna for leptin is elevated in the wat of obese humans and animals and blood levels also increase, since there is impaired leptin action called leptin resistance, leptin does not function sufficiently to suppress appetite or promote energy expenditure [18, 2022]. On the other hand, it is also known that leptin has inflammatory effects, such as increasing the expression of inflammatory cytokines involving tnf- by acting on monocytes . Evidence shows that seven weeks of spontaneous running tr reduces the expression of mrna for leptin in the visceral and subcutaneous wat of obese rats (table 1). Additionally, other research indicates that even a short duration (four weeks) of spontaneous activity reduces leptin mrna expression in rat wat (table 1). For obese humans, however, one study found that even 12 weeks of one - hour aerobic exercise sessions had no effect on the expression of mrna for leptin in subcutaneous wat (table 1). On the other hand, there have been many studies on the effects of tr on the human blood levels of leptin (table 2) [3450]. Many cases have shown that concentrations of leptin decrease with a reduction in wat mass (table 2) [34, 36, 4145, 47, 48]. By contrast, when no significant differences are observed in blood leptin levels after tr, neither is body fat reduced (table 2) [34, 35, 39]. Therefore, the reduced blood concentration of leptin after tr is due more to the reduction in body fat caused by tr than to the effects of tr itself . Some studies, however, suggest that a longer duration (12 weeks) of tr or tr with caloric restriction can contribute to a reduction in blood leptin concentration that is independent of the influence of body fat reduction (table 2) [34, 37, 40, 46]. Several studies have also concentrated on the effects of resistance training, such as the bench press exercise, on blood leptin levels (table 2). One study on postmenopausal obese women found that after performing three days a week of resistance training using machines and restricting diet for 16 weeks, blood leptin levels were decreased compared with pretraining levels, but that resistance training alone had no effect on leptin . However, when elderly individuals were divided into low intensity (4550% 1 repetition maximum [rm]), moderate intensity (6065% 1 rm), and high intensity (8085% 1 rm) groups and performed 60-minute exercise sessions three times a week for six months, blood leptin levels were lower in all the groups compared with the respective pretraining levels, and the magnitude of this decrease was significantly greater in the high intensity group than in the low and moderate intensity groups . Furthermore, although blood leptin levels were higher at six months after the end of training than immediately after the end of training, the levels remained significantly lower than their pretraining values in the high intensity group . Since the discovery that gene expression of the major inflammatory cytokine tnf- is elevated in wat in animal models of obesity, there have been many studies on its involvement in insulin resistance and its other actions [51, 52]. Expression of tnf- increases not only in the wat of obese animals, but also in that of obese humans; that is, tnf- has a strong positive correlation with body mass index (bmi) and blood insulin levels [5355]. Tnf- weakens insulin signaling by insulin receptor substrate 1-mediated inhibition of insulin receptor tyrosine kinase activity in areas such as skeletal muscle and causes reduced expression of glucose transporters and adiponectin in adipocytes, which contributes to the development of insulin resistance [5658]. There is no clear consensus regarding the effects of tr on tnf- in wat (table 1). For example, increased expression of tnf- in visceral wat in mice that became obese after six weeks of consuming a high - fat diet can be suppressed by spontaneous running [25, 26]. Additionally, our studies showed that nine weeks of treadmill running decreased the tnf- protein content of the rat wat [27, 28], but some results have shown a contrasting increase after tr [24, 29, 30]. Studies regarding obese individuals have also examined the effects of tr on tnf- expression in wat . Although one study found that tnf- expression in the subcutaneous wat of severely obese male and female adults decreased after 15 weeks of performing tr, such as walking for five days a week and undergoing diet therapy; a conflicting study on obese adults found that there was no change in tnf- mrna expression in subcutaneous wat even when weight or body fat decreased after 12 weeks of aerobic exercise [32, 33]. There are also conflicting results for the blood concentrations of tnf- (table 3) [33, 42, 45, 5963]. A study on diabetic patients showed that although there is no change in blood tnf- concentration after four weeks of dietary restrictions and walking tr in nonobese diabetic patients, the concentration decreased in obese patients . Furthermore, when obese adult women exercised on a bicycle ergometer for 30 minutes a day, five days a week at 70% vo2 max for 12 weeks, decreases in blood concentrations of both tnf- and soluble tnf receptor 2 were observed in both the women with insulin resistance and those without . In another study, however, a 15-week combination of diet therapy and tr did not affect the tnf- level in obese individuals . In yet another study, 12 weeks of endurance tr mcp-1, which is identified as a monocyte chemotactic factor, shows increased expression in the wat of obese mice, and elevated mcp-1 contributes to inflammatory changes by inducing macrophage infiltration into wat via its receptor, c - c chemokine receptor-2, which is expressed in monocytes and macrophages [70, 71]. In mice that are genetically modified to only express mcp-1 excessively in adipocytes, infiltration into visceral wat by macrophages is elevated when compared with control mice, and there is increased expression of macrophage markers and tnf- genes in the tissue, as well as increased insulin resistance [70, 71]. Mice that consume a high - fat diet show increased expression of mcp-1 mrna in visceral wat, but this expression is suppressed by six weeks of spontaneous running activity (table 1). Additionally, other studies where mice both consumed a high - fat diet and underwent treadmill running, mcp-1 mrna expression, which had increased due to the mice's high - fat diet, was reduced by tr (table 1). Moreover, nine weeks of treadmill running has reduced mcp-1 protein levels in rat subcutaneous and visceral wat (table 1). However, there were no changes in expression of mrna for mcp-1 either in the subcutaneous and visceral wat of rats that performed four weeks of spontaneous running or in the subcutaneous wat of obese humans who performed 12 weeks of aerobic exercise (table 1) [24, 32]. There seems to be consensus that tr diminishes blood levels of mcp-1 (table 3). The blood concentration of mcp-1 was reduced in rats by nine weeks of treadmill running tr . Studies on human patients with metabolic syndrome and obese individuals have also shown reductions and downward trends in mcp-1 after 12 weeks of tr . A 15-week combination of tr and diet therapy also reduced the blood concentration of mcp-1 in obese individuals . Adiponectin increases fatty acid oxidation and glucose uptake in skeletal muscle and inhibits gluconeogenesis in the liver [72, 73]. Adiponectin also inhibits the expression and secretion of tnf- in macrophages and increases the production of anti - inflammatory cytokines such as interleukin (il)-10 . Therefore, adiponectin is thought to have anti - inflammatory effects . In accordance with that function, the expression of mrna for adiponectin is reduced in the wat of genetically obese mice and obese humans, and both obese individuals and diabetic patients have a lower blood concentration compared with healthy individuals [75, 76]. Insulin resistance and hypertension are improved when kkay mice (mouse models of obesity and diabetes) are administered physiological concentrations of adiponectin, and insulin resistance is observed in ko mice deficient in adiponectin, suggesting that obesity - induced decreases in adiponectin expression in wat are closely associated with the development of insulin resistance and the onset of diabetes [72, 73]. A 15-week combination of tr and diet therapy or 12 weeks of aerobic exercise has shown increases in the expression of mrna for adiponectin in the subcutaneous wat of obese individuals (table 1) [32, 33]. In studies on rats, nine weeks of treadmill running has increased the mrna expression in visceral and subcutaneous adipocytes (table 1). In at least one study, short periods of consuming a high - fat diet increased adiponectin expression in the subcutaneous wat of rats, and tr by spontaneous running activity suppressed this increase . That study found no effect of tr on adiponectin mrna expression in visceral wat (table 1). As with leptin, there have been many studies on the effects of tr on the blood levels of adiponectin (table 4) [32, 33, 38, 42, 45, 50, 6569]. Although most indicate that there is no change, some studies show that it increases, so there is no consensus on this point . Hulver et al . Found that the blood concentration of adiponectin did not change after obese adults performed aerobic exercises such as running at 6585% vo2 max four times a week over a period of six months . Another study on diabetic men also found no change in the blood concentration of adiponectin after eight weeks of performing aerobic exercise three times a week, even though the amount of visceral fat decreased . Even after elderly obese men and women performed tr for 60 minutes on a treadmill or bicycle ergometer at 8085% of their maximum heart rate five times a week for 12 weeks, there was no change in the blood concentration of adiponectin despite the deceases in bmi and body fat . Contrary studies have found that 60 minutes of tr, such as running performed four times a week for four weeks, has led to increases in the blood concentration of adiponectin along with decreases in body fat in diabetics and individuals presenting impaired glucose tolerance . In a similar manner, the blood concentration of adiponectin has been increased along with reduced bmi and body fat mass after seven months of tr such as slope jogging and dumbbells performed four to five times a week in obese young women . Il-6 is a cytokine that has a variety of functions such as regulating hematopoiesis, immune response, and inflammatory response . This cytokine also is known to have anti - inflammatory effects, and may have both proinflammatory and anti - inflammatory properties [77, 78]. Diabetic and obese individuals have high blood concentrations of il-6, and its mrna expression is elevated in the subcutaneous adipocytes of individuals presenting insulin resistance . Furthermore, il-6 acts on adipocytes to inhibit insulin signaling [79, 80]. Many studies show that il-6 levels increase in response to acute exercise; for instance, a single bout of exercise has increased the blood concentration of il-6 more than 100 times . However, this increase in blood concentration was not due to increased production by wat, but rather by increased production in skeletal muscle, an organ that produces il-6 [78, 81]. A 15-week combination of tr and diet therapy reduces the expression of mrna for il-6 in the subcutaneous wat of obese individuals (table 1). However, although some studies show that the blood concentration of il-6 decreases after tr, other studies have shown no change, so yet again there is no consensus (table 5) [33, 42, 62, 63, 66, 68, 78]. The size of wat (adipocytes) greatly affects the expression of adipokines . As for leptin, mrna expression and secretion are positively correlated with the size of adipocytes isolated from rodents and humans [31, 8284]. Similarly, in isolated adipocytes of humans, secretion of tnf-, mcp-1, and il-6 is positively correlated with cell size, and after correction for the cell surface, there is still a significant difference between very large and small adipocytes for mcp-1 and il-6 . Nevertheless, mrna levels for tnf- show no significant correlation with mouse adipocyte volume . On the other hand, although the expression of adiponectin is reduced in the wat of genetically obese mice and obese humans, the mrna expression and secretion of adiponectin is positively correlated with isolated adipocyte size in rats and humans [31, 75, 76, 83]. One of the reasons for this discrepancy is speculated that reduced adiponectin expression in vivo may be the result of inflammatory adipokines, such as tnf-, rather than increases in the size of adipocytes . It is well known that tr reduces wat mass, and, therefore, the reduction of wat is thought to be a major factor in the effects of tr on adipokine expression in wat (figure 3). However, further research is needed regarding other effects of tr . Recently, an interesting study has examined the relationship between tr - induced changes in adipokine expression and wat mass . Christiansen et al . Divided obese subjects into a group that underwent 12 weeks of combined aerobic exercise and diet therapy and a group that underwent diet therapy only, and after adjusting weight loss to approximate amounts, found no difference in changes in either the expression of inflammatory - related adipokines in subcutaneous wat or in the circulating markers of inflammation; that is, tr seemed to have had no weight - independent effects in that study . On the other hand, when the authors observed a reduced level of leptin mrna and an elevated mrna level of adiponectin in rat visceral adipocytes after nine weeks of treadmill running, it suggested that the decrease in leptin mrna expression depended on a reduction in adipocyte size, and that the increase in adiponectin mrna was mediated by factor(s) other than adipocyte size . In addition, oberbach et al . Found that actual increases in blood adiponectin after tr were of a higher magnitude than increases in blood adiponectin levels that were predicted according to a regression line drawn from the negative correlation between body fat and the blood concentration of adiponectin . During exercise, the secretion of catecholamines from the adrenal medulla and sympathetic nerve peripheries breaks down triglycerides within the adipocytes . Several reports have indicated that -adrenoceptor agonists affect the expression of some adipokines, such as tnf- and adiponectin in wat . Administration of -adrenoceptor agonists in lean mice results in upregulation of tnf- and downregulation of adiponectin in epididymal wat [85, 86]. These findings seem to conflict with the beneficial effects of exercise on the disturbance of adipokines . Nevertheless, during exercise, since energy consumption is enhanced, the blockage of lipogenesis by the impaired insulin signaling in wat might play reasonable roles in the proper execution of exercise . In contrast to lean mice, -adrenoceptor agonists recovered the declined mrna expression of adiponectin and suppressed the overexpressed mrna level of tnf- in wat of kkay mice . Therefore, in obese and type 2 diabetic patients, it is likely that the secretion of catecholamines during exercise is one of the reasons for the attenuation of dysregulated adipokine expression in wat (figure 3). Various possible mechanisms besides decreased wat mass and secretion of catecholamines have been proposed, including decreased oxidative stress and improvement of hypoxia in wat (figure 3). Adipocytes have produced reactive oxygen, and obesity - induced increases in oxidative stress in wat may be a cause of the dysregulated expression of inflammatory - related adipokines . Studies have shown significantly lower levels of lipid peroxidation in wat around the epididymis and retroperitoneum of rats that had undergone tr compared with a control group, and elevated protein levels of the antioxidant enzyme manganese superoxide dismutase (mn - sod) in the epididymal wat of tr group rats [27, 28]. In that study, not only were protein levels of tnf- and mcp-1 significantly lower in the epididymal wat of the tr group, compared with those of the control group, the phosphorylation of extracellular signal - regulated kinase, which is activated by reactive oxygen and is important for the expression of mcp-1, also was reduced by tr in wat around the epididymis and retroperitoneum [27, 89]. Nevertheless, because acute exercise elevates oxidative stress in the body, the adaptation of wat against exposure to oxidative stress from exercise, in other words, the expansion of antioxidant systems via increases in mn - sod, could be one reason for decreased levels of proinflammatory adipokines . Recent evidence that tissue hypoxia is involved in obesity - induced inflammatory changes in wat has attracted the attention of researchers . In fact, oxygen partial pressure is lower in the wat of obese animals and humans compared with controls, and results show that this may be related to the inflammatory response in wat [91, 92]. Although some studies have focused on the impact of tr on blood flow in wat, results from those studies appear to indicate that when wat mass decreases due to tr, blood flow in the tissue increases . We found that expression of mrna for vascular endothelial growth factor and its receptor was elevated in the wat stromal vascular fraction cells of rats that had engaged in tr, and that the vascular endothelial cell count per unit area had increased . Thus, the increased blood flow to wat produced by tr eliminated the obesity - induced hypoxia in wat and could possibly have led to a weakening of the inflammatory changes in wat . In many of the previous studies that examined the effects of tr on adipokine expression in wat and on the blood levels of adipokines in human subjects, body mass, bmi, or wat mass reduction is observed (tables 15). For this reason, it remains unknown whether or not low - intensity tr that does not entail such reduction alters adipokine expression in wat or blood adipokine levels . As described in the previous chapter, adipokine expression is affected by the size of the wat (adipocyte). Among adipokines, expression of leptin seems to be especially largely affected by adipocyte size [8284]. In studies that examined the effect of tr on the blood leptin level in human subjects, results indicated that, in many cases, blood leptin levels do not change without a reduction in body fat; that is, decreased blood leptin levels are thought to be caused by exercise - induced wat mass reduction (table 2) [34, 36, 4145, 47]. In contrast, some studies have reported that the reduced blood leptin level shows beneficial effects of tr without wat mass reduction . For example, studies on adult males and females have shown that only the female subjects exhibit reduced blood leptin levels without body fat loss after undergoing 12 weeks of tr . Similarly, ishii et al . Have demonstrated that tr in type 2 diabetic subjects reduces serum leptin levels independent of changes in body fat mass . On the other hand, increased blood adiponectin level through tr is also accompanied by reductions in body mass, bmi, or wat mass (table 4) [33, 45, 48, 65, 66]. Although hsieh and wang observed that the blood adiponectin level was significantly elevated in type 2 diabetes patients who performed low - intensity tr (20 min / day, 5074% maximum heart rate) and adequate calorie restriction for one year, this particular study showed that body mass reduction seemed to be beneficial for increases in adiponetin . However, other reports indicate that blood adiponectin levels do not change if body mass is decreased [38, 42, 67]. Thus, it is difficult to conclude at this stage whether loss of body and/or wat mass is indispensable for adiponectin elevation . Moreover, the effects on tr - induced body and wat mass reduction may differ depending on the type of adipokine . Taken together, these results show that although further examination is necessary, it is conceivable that changes in adipokine expression in wat and blood adipokine level require tr that is sufficiently intense to reduce body mass or more specifically wat mass . Skeletal muscle is responsible for physical exercise, and it is the largest tissue in the body . Undernutrition, aging, and sickness cause a decline in skeletal muscle mass (a condition known as muscular atrophy), deteriorating one's exercise capacity [95, 96]. Moreover, skeletal muscle has a substantial impact on the overall metabolism of the body . For instance, skeletal muscles in patients with obesity and type 2 diabetes have reduced glucose metabolic capacity due to insulin resistance, and these observations are considered to be associated with the patients' clinical conditions . Many studies have shown that tr can increase mitochondrial proliferation and boost the expression of a glucose transporter 4 (glut4), and can in turn enhance lipid and glucose metabolic capacities [98100]. Among the molecules involved in exercise - induced enhancement of glucose / lipid metabolic capacity in skeletal muscle, amp - activated protein kinase (ampk) and peroxisome proliferator - activated receptor- coactivator-1 (pgc-1) ampk is an enzyme that is activated when atp is converted to amp and is a sensor of energy status that maintains cellular energy homeostasis [101, 102]. Skeletal muscle ampk is activated by muscle contraction, treadmill running, and stimulation by its agonist aminoimidazole carboxamide ribonucleotide (aicar). Upon activation glucose uptake elevation has been associated with the induction of glut4 translocation to the cell membrane [106108]. Ampk activity has also been reported to be involved in fatty acid uptake through the fatty acid translocase fat / cd36 and fatty acid oxidation mediated by reduced acetyl - coa carboxylase enzymatic activity [103, 109]. Tr has been shown to enhance both expression and activation of ampk in skeletal muscle, and chronic ampk activation in skeletal muscle can increase the number of mitochondria even in the absence of tr, suggesting that tr - induced ampk activation is strongly involved in the increase in the mitochondria of skeletal muscle [110, 111]. However, a conclusion is yet to be drawn because ampk ko mice that underwent tr also showed increases in skeletal muscle mitochondria . Transcription coactivator pgc-1 forms a complex with nuclear receptors and transcription factors to regulate gene transcriptions, or more specifically, expression of genes involved in mitochondrial biosynthesis [113115]. In fact, mice with pgc-1 overexpression showed (1) increased number of mitochondria, (2) enhanced expressions of oxidizing enzymes such as cytochrome oxidase in skeletal muscle, and (3) transition to type i muscle fibers [114, 115]. Physical exercise increases pgc-1 transcription and potentially pgc-1 activity through posttranslational modifications, and concomitant pgc-1-mediated gene regulation is suggested to be an underlying mechanism for adaptations in skeletal muscle, when exercise is repeated . Therefore, increases in mitochondria and increased insulin sensitivity in skeletal muscle by endurance tr are thought to dramatically impact the energy consumption of the whole body . Moreover, enhanced glucose / lipid metabolism in skeletal muscle is considered to be indirectly involved in wat reduction, which results in altered adipokine expression (figure 3). Additionally, because resting metabolic rate (rmr), which is the largest component of the daily energy budget in most human societies, is reportedly elevated owing to both aerobic and resistance training in human subjects, although some studies have failed to find such an effect, enhanced rmr is likely to cause alteration of adipokine expression following wat mass reduction due to increased energy expenditure in the resting state (figure 3). Nevertheless, the detailed mechanisms and whether mediators, such as myokines, from skeletal muscle act on the existence of wat remain unknown . On the other hand, it is interesting that evidence is mounting on the new effects of adipokine on skeletal muscle metabolic capacity . Recent observation of ko mice showed that a lack of adiponectin receptor in their skeletal muscle showed a reduced mitochondrial content, reduced type i muscle fibers, and decreased capacity for exercise, suggesting that adiponectin is involved in mitochondrial biogenesis in skeletal muscles . Furthermore, there is a significant positive correlation between blood adiponectin level and ampk activity in the lateral great muscles in men . In the future, it is crucial to examine the effect of tr on adipokine expression not only in wat alone but also in terms of cross - talk between wat and other tissues involving skeletal muscle . Although reports on the effects of exercise on adipokine levels in wat and blood may not always agree due to differences in experimental subjects, exercise intensity, or exercise duration, it is reasonable to believe that there is at least a positive effect . Although tr - induced wat reduction is one of the key reasons for attenuation of dysregulated expression of adipokines, detailed studies about not only wat - reducing effects of tr but also other effects, such as antioxidative effects and angiogenic effects, will be necessary to show the usefulness and distinctiveness of tr . Furthermore, it may be significantly beneficial to examine the cross - talk between wat and other tissues involving skeletal muscle and to what degree wat contributes to tr - induced changes in blood adipokine levels . Because the importance of exercise as a tool for preventing and improving obesity and lifestyle - related diseases can be expected to grow in the future, further research is desirable.
Although endovascular aneurysm repair (evar) of intact infrarenal abdominal aortic aneurysms (aaa) has been associated with a reduction in early mortality and morbidity as compared with open repair, the benefits of an endovascular strategy do not appear to translate to the treatment of ruptured aneurysms . Two randomized trials have demonstrated no difference in 30-day mortality when comparing open and endovascular repair of ruptured aaa . The nellix endovascular aneurysm sealing (evas) system (endologix, irvine, ca, usa) offers a new approach to the endovascular treatment of aaa, achieving aneurysm exclusion by sealing the aneurysm sac . Each nellix system consists of two identical catheter - based devices with a 10-mm flow lumen being created by two balloon - expandable polytetrafluoroethylene covered cobalt the stents are mounted on balloons for deployment and are surrounded by polyurethane endobags . While conventional endografts have a proximal and distal attachment zone for fixation, the evas device fixes the two stents within the aneurysm sac using the endobags, which are filled with a polyethylene glycol (peg)-based hydrogel that conforms to the aneurysm flow lumen and solidifies within minutes of delivery, providing fixation and seal at the aortic neck and iliac arteries . Endobag filling is performed under pressure monitoring, which allows the polymer to be instilled to a pressure of 180 to 220 mm hg, which will seal the aneurysm . One of the potential difficulties in treating ruptured aneurysms with evas is whether the aneurysm rupture site will preclude the development of an adequate pressure during polymer fill to enable aneurysm sealing . Moreover, there is a theoretical risk of increasing the size of tear at the site of rupture . Adverse aneurysm morphology remains a key challenge for conventional evar, and many devices are currently used outside the manufacturer s instructions for use (ifu), according to clinical need . The ifu accompanying the nellix device suggest that an endovascular strategy may be feasible in a greater number of patients with a broader range of aortic morphology . We present our initial experience using evas in the treatment of patients with a ruptured infrarenal aneurysm . Prior to december 2013, 50 patients with aaa were treated with nellix in an elective setting at our institute . All surgeons treating patients in the cases presented here had experience in 10 elective cases using the nellix device . Eligibility for evas in infrarenal ruptured aaa was established based on morphological suitability and the decision of clinicians with audited experience in emergency evar . Ruptured aaas with morphology within the ifu for standard evar devices were not considered for evas . Between december 2013 and march 2014, 5 patients (median age 71 years, range 5790; 3 men) with ruptured infrarenal aaas were treated with evas . All patients were conscious and lucid during the consent process and the novelty of this treatment option was explained to each before they provided written informed consent prior to aneurysm repair . Baseline characteristics, aneurysm morphology, and procedural details for each of the patients are displayed in table 1 . Aneurysm morphology in 4 of the 5 patients was noncompliant with the ifu for both nellix and standard evar devices; the remaining patient was outside the ifu for standard evar devices but treated with nellix under standard ifu for the nellix system (table 1). Prospectively collected baseline clinical and morphology data included the hardman index and aneurysm neck length, diameter, and angulation (table 1). The decision to perform evas under local or general anesthesia centerline measurements on computed tomographic angiography (cta) reconstructions were used to calculate aneurysm length and volume prior to surgery to allow stent sizing and thawing of the appropriate volume of polymer . Local anesthesia was used in 4 cases, but 1 patient required general anesthesia (table 1). Bilateral percutaneous access via the femoral arteries with ultrasound guidance was preferred, with proglide closure devices (abbott vascular, abbott park, il, usa) deployed at both access sites . However, a unilateral cutdown was required in 2 patients where percutaneous closure application failed . After visualization of the lowest renal artery under angiographic control, a calibrated pigtail catheter was used to confirm the length to the bifurcation and subsequently the length of the devices needed . The median stent length was 160 mm (range 140180) on the left side and 170 mm (range 140180) on the right (table 1). The nellix devices were inserted through the femoral access sites bilaterally and advanced over wires into position at the renal ostia . After retracting the covering sheaths, the stent - grafts were deployed by simultaneous inflation of the balloons within the stents to 7 atm (figure 2a). The endobag was inflated first with a saline pre - fill to confirm the required polymer volume then with the aqueous peg - based polymer (figure 2b). Five minutes following polymer curing, endobag pressures were checked again, and the delivery devices were removed . The stents were routinely postdilated using a 10-40-mm angioplasty balloon (cook medical, bloomington, in, usa) inflated to 7 atm . Completion angiography was used to demonstrate successful sealing of the ruptured aneurysm with bilaterally patent stent - grafts and iliac arteries . If the angle between the nellix stent - graft and the iliac vessel wall was too large or ended at a curvature within the iliac artery, additional uncovered self - expanding stents (cook medical) were applied . In these 5 cases, a single adjunctive bare metal stent was deployed at the distal end of the evas device (iliac arteries) in 2 patients . A median of 3 units of blood was required during surgery, which lasted a median 85 minutes (interquartile range 7590). Median intensive care unit stay was 3 days (range 06) and median hospital stay was 8 days (range 040) (table 1). (a) the nellix stents deployed within a ruptured infrarenal aneurysm, with the endobags not insufflated . (b) the nellix device fully deployed with hemostatic control of the rupture and perfusion to both limbs . Postoperative surveillance consisted of a predischarge duplex scan and cta if postoperative renal function permitted; cta was repeated at 6 months after discharge, and the duplex scan was done at both 3 and 6 months in an outpatient setting (figure 3b). The surveillance requirements of evas remain to be defined, and this protocol differs from the duplex - only surveillance conducted following ruptured aaa repair with a conventional endograft . (a) preoperative axial computed tomography angiography (cta) scan of the aorta showing the hematoma surrounding the aneurysm, indicative of rupture . (b) the postoperative axial cta with the endobags and stent - grafts within the aneurysm . Among the 5 patients treated with this device, 2 died . Hemodynamic parameters deteriorated further following anesthetic induction; an endovascular sealing system was deployed, but the patient experienced cardiac arrest on the operating table and died . The other patient had hemodynamic instability on postoperative day 2; an emergency laparotomy was performed, and bleeding was observed from the aneurysm sac, assumed to result from a proximal type i endoleak . Postoperative cta performed in the 3 surviving patients demonstrated no endoleak and no limb occlusions . Median follow - up of surviving patients was 9.3 months; at 3 and 6 months, all 3 devices were patent on duplex ultrasound and cta, with no evidence of endoleak or insufficient aneurysm sac sealing among the survivors . This report summarizes a preliminary clinical experience with the nellix device for ruptured aaa repair . In contrast to evar devices, the nellix system uses a sac - anchoring endoprosthesis for aneurysm exclusion . The small numbers in this report do not permit comparison with existing evar devices or open repair; however, they do support the feasibility of evas for ruptured infrarenal aneurysm repair in cases with morphological suitability, for example, those with a maximum aortic flow lumen diameter of 60 mm . Evas may therefore provide an alternative to open or conventional endovascular repair of ruptured aaa . The nellix system offers several potential advantages over a conventional endografts for ruptured aneurysm repair . First, evas has been shown to be applicable to a wider range of aortic morphology (figure 4) than currently available evar devices . The potential to use evas to treat patients who are noncompliant with the ifu of existing aortic stent - grafts is attractive in light of the higher incidence of sac expansion observed in patients treated by evar outside the ifu . The ifu for the nellix devices specify that they are not suitable for patients with small or large common iliac arteries (<8 or> 35 mm, respectively) or those with a large patent flow lumen (> 60 mm) within the aneurysm sac . Preoperative computed tomographic angiography reconstruction showing hostile neck anatomy amenable to treatment with endovascular aneurysm sealing (evas). The inventory of devices required for evas is less compared with that for conventional endografts, as the only sizing variation in the nellix system is related to stent length . Preoperative sizing is therefore straightforward, involving the selection of only a suitable length of device, which may be of benefit in an emergency setting . The avoidance of contralateral limb cannulation in evas may reduce the overall procedure duration in certain cases . The nellix system also provides an option for early hemostasis through endobag inflation with saline, while maintaining limb perfusion . A final advantage is the avoidance of type ii endo - leaks with aneurysm sac sealing . The potential disadvantages of the evas technique for ruptured aneurysm repair include a risk of enlarging a rupture or tearing the aorta with the pressure used to fill the endobags . In addition, there is the potential for failure to seal a ruptured aneurysm due to an inability to generate the required pressure within the ruptured aneurysm sac . It is suspected that one patient in this cohort developed a type i endoleak that was not obvious on surveillance ct performed on postoperative day 1 . Despite satisfactory appearances on the cta, the patient was returned to surgery for laparotomy during a period of hemodynamic instability, where bleeding was observed from the aneurysm sac . As such, it would be reasonable to assume that a type i endoleak was responsible for reperfusion of the sac . On retrospective review of the postoperative images, there was opportunity to increase the proximal sealing zone by deploying the stent closer to the renal ostia . The detection of endoleaks with cta in the early postoperative period after evas does pose a challenge due to the opacification of the polymer - filled endobags . Although preliminary, the current report of 5 patients provides the most substantial information to date about the feasibility of evas in ruptured aneurysm repair . The technique appears feasible, but clearly a refined protocol for the use of evas in ruptured aaa is required with consideration of the need for saline pre - fill and target polymer fill pressures in addition to identifying specific morphological parameters that predict success or failure . In each patient there were several reasons for not selecting open repair, including previous laparotomy or peritonitis associated with continuous ambulatory peritoneal dialysis . In other cases, the patients were considered high risk for general anesthesia based on comorbidity, and a local anesthetic alternative was considered optimal . However, open repair should not be forgotten as a treatment option for ruptured aaa . An important consideration with any new device is durability, and longer follow - up is needed in a cohort of patients treated with evas to compare this modality with existing endovascular techniques . Endovascular aneurysm sealing appears feasible for the management of ruptured aneurysms, with acceptable short - term outcome, albeit in a small series of patients . The application of evas devices for ruptured aneurysm repair may broaden the selection criteria of the current endovascular strategy to include patients with complex aneurysm morphology.
The prevalence of chronic diseases is increasing drastically, and is predicted to increase over the next two decades (1). There is a dramatic re - emergence of old infectious diseases like tuberculosis and cholera, and appearance of new ones, like hiv and hepatitis c (2). Integrated preventive care, rather than treatment, is more effective in tackling the growing burden of both infectious and chronic diseases . In order to generate these preventive measures, cause of illness and although these precautionary measures are very effective, but they are difficult to achieve because health is influenced by a number of related factors, all of which need to be coordinated to implement and adopt preventative policies . Previously, it was thought that occurrence of a disease could be explained through bio - medical model that assumed that disease is caused by a specific etiological agent inside the body (3), but now health behavior, environment, socio - economic status, and genetic factors are also considered to be closely interrelated to health along with biological factors (4). The social - determinants - of - health school and the health field concept are important continuations of these health theories (5, 6). Infectious diseases are not an exception for these disease models (7), and personal behavior, environment, and socio - economic status are closely related to many infectious diseases (8), e.g., the prevalence of diarrhea and cholera are influenced by clean water, sanitation, and proper hygiene . This transition regarding determinants of health raised scholars interest in public views on health and its determinants (9), and this enhanced centrality of public views has become the norm in health policy as well (10). Previous studies have either focused on health and its determinants in scientific view or just stressed the significance of public opinion for policymaking (9, 11). However, few efforts have been made to evaluate the concept of lay people regarding determinants of health and factors affecting it . People belonging to different social status, education level, and health state have different perceptions regarding health (12) which further influence their health behaviors . There is little point in developing health plans with - out understanding how people s belief about health are affected by their individual circumstances, thereby influencing the take - up of services (13). Up to our knowledge prior studies which assessed public opinions and beliefs regarding illness limited their focus to only one specific disease, like obesity or hypertension (14, 15) also ignoring the factors effecting people s attitude . Furthermore, these studies were done at national level, representing the situation in one country only . Therefore, our study aimed to evaluate the general (not disease specific) perception of lay people regarding determinants of health at a global level and to identify the effect of various social and demographic factors on public believes . This might help policy makers in developing a good concept of preferences and misconceptions of health among lay people and in designing and implementing reform proposals that can influence their behavior, thus helping to control the disease burden throughout the world . To achieve these goals, we performed a multilevel analysis on data from 29 countries, most of which are high - income countries . Issp is a continuing annual program of cross - national collaboration based on voluntary participation of countries . The topic of 2011 survey was health and health care, and it included data from 29 member countries . Issp use a standardized questionnaire for survey and all the variables have a validated scale for assessment . Sampling procedure differs for the individual countries like partly simple and partly multi - stage stratified random sampling . Mode of interview also differs for the individual countries including face - to - face interviews, postal survey and web survey (17). The data for gni and gini was collected from the world bank website (18). Total sample size was 45,563 while sample size for each country is shown in table 1 . Names of countries with sample size and variable data names of 29 countries included in analysis along with population size of each country and% of smokers, employed and obese people by country is shown four indexes were constructed in order to capture public perspectives on the determinants of health . The 2011 issp question linked to these indicators is people suffer from severe health problems how much do you agree or disagree with each of the following items? A) because they behaved in ways that damaged their health, b) because of the environment they are exposed to at work or where they live, c) because of their genes, d) because they are poor . Three dummy variables were assigned for age as younger (1939 yr . ), middle aged (4059 yr . ), and older (60 yr . And above). Average monthly household income, gni and gini variables were changed into a quartile index (scaled 1 to 4, where 4= above the third quartile). Four education dummy variables were assigned, i.e., elementary school (06 years of education: reference group), junior high school (79 years of education), senior high school (1012 years of education), and post - secondary school (13 or more years of education). A continuous variable of insurance coverage was introduced (scaled 13 where 3= covers very well). Self - rated health status (q26: scaled 1 to 5 where 5=very good), bmi (q28, 1=overweight or obese, 1 is yes), drinking and physical activity (q25a and 25b: scaled 15 where 1=never and 5=daily). Total number of participants was 45,563 and sample size ranged from 936 to 3,319 among countries . We constructed a box plot by frequency analysis to see the percentage of agreement of people with different determinants of health using ibm spss 19 software . Chi square test was performed using spss, and a bar graph was generated to analyze trends in the association between agreement with determinant of health and gni quartiles by using microsoft excel . Only score 4 and 5 (agree and strongly agree) was used to find percentage of agreement with each health determinant . Multilevel logistic regression model with individuals as first level and countries as second level was used . A single - model strategy was employed to run the analysis using stata 12.0 software . The model included all the independent variables and was analyzed separately according to the four dependent variables to obtain the results of the regression . We applied an individual weighting factor during analysis to adjust the distribution of primary sampling unit and to correspond with that of each nation s population . No ethical approval was needed for this study because we used already assembled data from issp and world bank . Informed consent is taken from all the respondents before survey by issp team (16). Issp is a continuing annual program of cross - national collaboration based on voluntary participation of countries . The topic of 2011 survey was health and health care, and it included data from 29 member countries . Issp use a standardized questionnaire for survey and all the variables have a validated scale for assessment . Sampling procedure differs for the individual countries like partly simple and partly multi - stage stratified random sampling . Mode of interview also differs for the individual countries including face - to - face interviews, postal survey and web survey (17). The data for gni and gini was collected from the world bank website (18). Total sample size was 45,563 while sample size for each country is shown in table 1 . Names of countries with sample size and variable data names of 29 countries included in analysis along with population size of each country and% of smokers, employed and obese people by country is shown four indexes were constructed in order to capture public perspectives on the determinants of health . The 2011 issp question linked to these indicators is people suffer from severe health problems how much do you agree or disagree with each of the following items? A) because they behaved in ways that damaged their health, b) because of the environment they are exposed to at work or where they live, c) because of their genes, d) because they are poor . The coding of answers is scaled from 1 to 5 where 5=strongly agree . Three dummy variables were assigned for age as younger (1939 yr . ), middle aged (4059 yr . ), and older (60 yr . And above). Average monthly household income, gni and gini variables were changed into a quartile index (scaled 1 to 4, where 4= above the third quartile). Four education dummy variables were assigned, i.e., elementary school (06 years of education: reference group), junior high school (79 years of education), senior high school (1012 years of education), and post - secondary school (13 or more years of education). A continuous variable of insurance coverage was introduced (scaled 13 where 3= covers very well). Self - rated health status (q26: scaled 1 to 5 where 5=very good), bmi (q28, 1=overweight or obese, 1 is yes), drinking and physical activity (q25a and 25b: scaled 15 where 1=never and 5=daily). Total number of participants was 45,563 and sample size ranged from 936 to 3,319 among countries . We constructed a box plot by frequency analysis to see the percentage of agreement of people with different determinants of health using ibm spss 19 software . Chi square test was performed using spss, and a bar graph was generated to analyze trends in the association between agreement with determinant of health and gni quartiles by using microsoft excel . Only score 4 and 5 (agree and strongly agree) was used to find percentage of agreement with each health determinant . Multilevel logistic regression model with individuals as first level and countries as second level was used . A single - model strategy was employed to run the analysis using stata 12.0 software . The model included all the independent variables and was analyzed separately according to the four dependent variables to obtain the results of the regression . We applied an individual weighting factor during analysis to adjust the distribution of primary sampling unit and to correspond with that of each nation s population . No ethical approval was needed for this study because we used already assembled data from issp and world bank . Informed consent is taken from all the respondents before survey by issp team (16). Among 45,563 participants out of 29 countries, 47% of participants were male and 52.7% were female . Table 1 shows the names of countries included in the analysis, along with their sample size, gni, gini, sex ratio, and average age . Percentage of agreement with different causes as determinants of health among different countries is shown in table 2 . Percentage of agreement with determinants of health by country percentage of people who agreed with each factor (health behavior, environment, poverty and genes) as determinant of health by country are shown frequency analysis showed that environmental factors had the highest percentage of agreement as determinant of health followed by genes, health behavior, and poverty respectively (fig . Total% of agreement with determinants of health chi square test revealed that the percentage of agreement with health behavior, environment, and poverty as health determinants increased with decrease in gni quartile, but no specific trend was observed for genes (fig . 2).% of agreement with different determinants of health according to gni quartiles beta coefficients and 95% confidence interval obtained from multilevel regression analysis are shown in table 3 . There was a significant negative association of females with health damaging behavior and positive association with environment and genes as health determinants . Elderly people had a positive relation with poverty while all the education dummies were related negatively with health damaging behavior as determinant of health . Multilevel models for dependent variables average income showed a negative association to environment and poverty along with a positive relation to genes, while employment status had a negative relation with health - damaging behavior and positive relation with environment and poverty . Gni was negatively related to environment and poverty whereas people with good insurance coverage agreed that poverty was a health determinant . Health damaging behavior was positively associated with self - related health status while environment and poverty were negatively related to it . Drinkers had a negative relation with environment and physical activity was associated positively with health behavior as health determinants . Among 45,563 participants out of 29 countries, 47% of participants were male and 52.7% were female . Table 1 shows the names of countries included in the analysis, along with their sample size, gni, gini, sex ratio, and average age . Percentage of agreement with different causes as determinants of health among different countries is shown in table 2 . Percentage of agreement with determinants of health by country percentage of people who agreed with each factor (health behavior, environment, poverty and genes) as determinant of health by country are shown frequency analysis showed that environmental factors had the highest percentage of agreement as determinant of health followed by genes, health behavior, and poverty respectively (fig . Total% of agreement with determinants of health chi square test revealed that the percentage of agreement with health behavior, environment, and poverty as health determinants increased with decrease in gni quartile, but no specific trend was observed for genes (fig . Beta coefficients and 95% confidence interval obtained from multilevel regression analysis are shown in table 3 . There was a significant negative association of females with health damaging behavior and positive association with environment and genes as health determinants . Elderly people had a positive relation with poverty while all the education dummies were related negatively with health damaging behavior as determinant of health . Multilevel models for dependent variables average income showed a negative association to environment and poverty along with a positive relation to genes, while employment status had a negative relation with health - damaging behavior and positive relation with environment and poverty . Gni was negatively related to environment and poverty whereas people with good insurance coverage agreed that poverty was a health determinant . Health damaging behavior was positively associated with self - related health status while environment and poverty were negatively related to it . Drinkers had a negative relation with environment and physical activity was associated positively with health behavior as health determinants . Our study shows that the highest percentage of people agreed with the work and living environment as determinant of health . Previously, general public believed that access to health care and personal behavior were the strongest determinants of health (19), but our results suggest that now lay people are well aware that other social factors, like environment can also influence their health . This increased public awareness can be attributed to larger media campaigns (20) as well as to an increase in environmental disease burden . An estimated 24% of global disease burden and 23% of all deaths are attributable to environmental factors (21). Our results also show that more than 60% of people agreed that genes and health behavior are determinants of health . Health behavior has a strong influence on health outcomes, and factors like smoking, alcohol and improper hygiene (infectious diseases) kill millions of people globally (22, 23) while genetic factors are also closely related to many chronic diseases, like copd and dementia, contributing to the disease burden throughout the world (24). Poverty is also an important risk factor to health, and many evidences support a link between poverty, malnutrition, and poor health (25). In our analysis, more than 50% of the respondents agreed that poverty is a determinant of health suggesting that even in high - income countries; people are sensitive to this issue . All these findings propose that common people are now getting aware of the broadened concept of health, and their thoughts are becoming consistent with new paradigm of social determinants of health model . Our analysis suggests that people with different socio - demographic characteristics have different perceptions about determinants of health . According to our findings, men agreed more with health - damaging behavior while women agreed more with environment and genes as determinants of health . As men are more exposed to socio - economic stress, the prevalence of unhealthy behavior like smoking and unbalance diet is more common in them (26), resulting in their perception of health behavior as cause of illness while women are mostly more health and diet conscious and instead of health behavior other social and environmental factors are more important to them as health determinants (27). Females also agreed more with genetic causes than males possibly because most genetic disorders are congenital and are diagnosed during pregnancy or immediately after birth (28), increasing the physical and psychological suffering of mother along with baby (29) resulting in women becoming more conscious and aware of genes being a health determinant . Most people suffer from chronic diseases with advancing age, resulting in an increase in their medical expenses (30). Thus, they cannot afford the high cost of medical treatments, which make them sensitive to poverty as a determinant of health . Among the education variables, there was a significant negative association of educated people to health - damaging behavior as determinant of health . Previous studies suggest that as education level of people increase, their knowledge is broadened, and they understand that health is no longer limited to individual behavior but is rather related to other social, economic, and environmental factors (31) while people who are uneducated have a limited focus and they consider only personal behavior to affect health, being unaware of contemporary concepts of health and its determinants . People with low - income agreed with environment and poverty while high - income people agreed with genes as determinant of health . Low - income people are usual victim of poverty, and poverty is strongly interrelated to many chronic and infectious diseases (32). Additionally, work and living environment of poor people increase the risk of illness and disability due to, for example, lack of access to clean water and sanitation and an increase in accidents and injuries at the workplace (25). All these factors contribute to perception of low - income people for environment and poverty as health determinants . Many previous studies have proven that high - income individuals are well - educated (33) and have better concepts about health than low - income individuals have . Therefore, high level of education of rich people may be the cause for their agreement with genes as determinant of health, ignoring environment and poverty, which are controllable factors for them . With respect to employment status, unemployed individuals agreed more with health damaging behavior while employed people agreed more to environment and poverty as health determinants . Many previous studies have suggested that unemployment is associated with poor health behavior and adverse health outcomes (34) while employed people are more exposed to mental stress and to hazards of their work environment (35) and are more sensitive to economic changes and poverty, resulting in difference of opinion on health determinants among them . There was an increase in percentage of agreement with health behavior, environment, and poverty as determinants of health with a decrease in gni quartiles while no specific trend was observed for genes, as health determinant (fig . 2). There was a significant negative relationship of environment and poverty to gni . This may be due to the fact that people living in a country with high gni usually have better work and living environment (36) and are less exposed to poverty and its related risk factors unlike people of low gni countries who are victims of poverty and are exposed to more hazardous work and living environment that expose them to poor hygiene, improper sanitation, and unsafe drinking water (25). Our analysis has revealed a significant positive association of insurance coverage to poverty as a health determinant . Many previous studies have proved that people with proper health insurance coverage have easy access to health care and preventive services and have better health outcomes (37), making them aware of insurance benefits and convincing them to think poverty as determinant of health which hinder poor people to afford proper health insurance . Individuals with good self - related health status agreed that health - damaging behavior was a determinant of health while individuals with poor self - reported health status and those with chronic illness agreed to environment, genes, and poverty . Individuals who report good health usually have good functional abilities and better health outcomes (38) that may be due to their healthy lifestyle thus, suggesting that they are well aware of the impacts of their behavior on health while people with poor self - related health may not be adopting healthy behavior and will attribute the cause of their illness to other uncontrollable factors . Additionally many previous studies have suggested that chronically ill people with poor health are considerably more likely to be poor, either due to their low income or increased medical expenditures (39), making them more sensitive to poverty as health determinant . As discussed earlier many chronic diseases have a genetic etiology (24), and some people with chronic illness may have a misconception about its genetic origin (40) thus, convincing the ones with poor self - reported health and with chronic illness for genes to be the determinant of health . Among health behavior variables, non - smokers and physically active people agreed that health behavior was determinant while smokers agreed that environment and poverty were determinants of health . This finding may be because non - smokers and physically active people are well aware of the adverse effects of smoking and sedentary lifestyle (41, 42). Thus, they are more likely to believe that health behavior is determinant of health . On the other hand, smokers are either unaware of the risk factors of smoking to their health (41) or may underestimate the magnitude of these risk factors (43). Furthermore, many studies have reported that smoking prevalence is more common among poor people with low socio - economic status (44) who is also exposed to hazardous work and living environment consequently reinforcing their belief that poverty and environment are health determinants . On the contrary alcoholism is mostly associated with antisocial behavior and other personality disorders (45), which make alcoholics ignorant of their surrounding environment and of its effects on their health . Firstly, we have presented a general concept of people regarding health determinants, but they may have different perceptions on the origin of specific diseases . However, we did not completely ignore disease - specific focus and added some covariates, like chronic illness, disability, and health status . Secondly, this study has adopted a global approach and all the countries are high - income countries except philippines, which is a low middle - income country according to world bank classification . Thirdly, these countries are not selected randomly but are based on voluntary participation in issp survey, which make the external validity uncertain for even high - income countries . Further exploratory studies are needed to clarify the public opinion regarding health and its determinants with focus on individual countries . This can help in policymaking and implication according to particular situation in a specific country . Despite these limitations,, we have focused on the perceptions that lay people hold on the determinants of health . Second, along with health behavior we have also analyzed the awareness of lay people about new emerging concept of other social determinants of health . Third, a number of social, economic, and demographic characteristics have been taken into account to observe their effects on public perception regarding health . Fourth, this study includes data from 29 countries of the world, which increase the generalizability of our results . People are now becoming aware of a broadened concept of health, and they now understand that their health can be affected by a number of surrounding economic and environmental factors . Our study also indicates that people belonging to different social, demographic, and economic backgrounds have different perceptions regarding determinants of health, which subsequently affect their attitudes and health behavior . In order to develop health education programs and preventive services that are compatible with individuals beliefs, policy makers must be able to identify and understand the specific factors that influence the individuals perception and behaviors with respect to health . Education programs and materials that are customized to address the unique needs and concerns of specific patients have shown promise in changing a range of health related behaviors (46, 47). People are more likely to thoughtfully process information when they perceive it to be personally relevant . Thus, tailored health programs and policies that address an individual s specific problems and concerns are more likely to stimulate change in behaviors and attitudes . Therefore, the result could be an increase in the uptake of preventive services and a decrease in disease burden . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc .) Have been completely observed by the authors.
Studies found lateral canals in 45% of 74 teeth1 and 27.4% of 1140 teeth observed.2 lateral canals are difficult to instrument and to irrigate during endodontic therapy and may allow bacterial growth.1 although some authors found no correlation between unfilled lateral canals and inflammation of the periodontal ligament,3 other studies demonstrated their potential pathogenicity after healing of periradicular lesions in relation with full filling of lateral canals.46 persisting bacteria in teeth endodontically treated7 may be located in uninstrumented areas like lateral canals.6 in this case, the three - dimensional obturation of the root canal system becomes extremely important, as it could prevent reinfection8 and isolate microorganisms in inaccessible areas, without access to space and nutrients.9,10 the capability of an endodontic filling technique to ensure the filling of thin and irregular ramifications is an important clinical parameter and may represent a favorable aspect of the filling technique . Different techniques have been proposed over the years, and several in vitro models have been proposed to compare the results of these filling techniques . For this purpose, considering the high number of in vitro techniques proposed over the years, a model with a main and various lateral canals would be an important tool with which to investigate and compare filling techniques.11 the purpose of this study is to compare the ability of three filling techniques to fill simulated lateral canals (continuous wave of condensation, thermomechanical compaction, and lateral condensation) and, also, compare the percentage of lateral canals filled by gutta - percha and sealer . Thirty extracted human single - rooted teeth were used for this study . All teeth presented extracted indications of advanced periodontal disease . A single operator carried out all steps . Conventional endodontic access was prepared using round diamond drill (kg sorensen, so paulo, brazil). The canal length was visually established by placing a size 10 k - file in each root canal until it was seen emerging through the apical foramen . The coronal and middle thirds of each canal were flared using 4, 3, 2, gates glidden drills (dentsply maillefer, ballaigues, switzerland). All teeth were instrumented to the working length of a size 40 k - file . A step - back preparation with sizes 45, 50, and 55 k - file was performed . After each instrument, the canals were irrigated with 3 ml of 2.5% sodium hypochlorite solution (biodinmica qumica, ibipor, paran, brazil) using a 27-gauge needle (hypodermic, shanghai, china) that penetrated to the middle third . The smear layer of the root canals was removed using irrigation with 10 ml of edta 17% (biodinmica qumica, ibipor, paran, brazil) through manual activation with a 40 k - file for 3 minutes for each canal, followed by a 5ml flush with sodium hypochlorite . After that, canal patency was verified using a size 20 k - file and dried by paper points (dentsply maillefer, ballaigues, switzerland). Three simulated lateral canals were then drilled on the mesial surfaces of the root (one in each third), perpendicular to the main canal at 3 mm, 6 mm, and 9 mm from the apex, using a #15 engine reamer (h. shein, new york, ny). The lateral canals showed a slightly tapered shape, with the base side to the external root surface (figure 1a). For all groups, grossman sealer (endofill, dentsply maillefer, petrpolis, rj, brazil) was used as the root canal sealer . The sealer was mixed according to the manufacturer s instructions and applied by coating the canal walls using the main cone itself . Afterward, the root canals were filled (figure 1b) according to following techniques . A medium gutta - percha cone 0.06 (konne ind . Ltda, belo horizonte, brazil) was fitted to working length using a calibrator rule (dentsply maillefer, petrpolis, rj, brazil) that adjusts the tip of the cone to the size 40 k - file . The heat source was activated, and a preheated medium plugger (system b, analytic technology, glendora, ca, usa) was inserted into the root canal to thermoplasticize and compact the gutta - percha at the apical third, 5 mm from the apex . Vertical compaction was performed using a size 2 plugger (jr instrumental ltda, belo horizonte, brazil). The middle and coronal thirds of root canal were backfilled using obtura ii (obtura corporation, penton, missouri, usa). A medium gutta - percha cone 0.06 was adjusted in the same manner as in the previous group . Lateral condensation was performed using a size c finger spreader (dentsply maillefer ballaigues, switzerland) and fine accessory gutta - percha cones . The gutta - percha excess was removed using a heat plugger, and the gutta - percha mass was thermomechanically compacted at the coronal and middle thirds of the root canals using a size 45 gutta - condensor (dentsply maillefer, ballaigues, switzerland) rotated at 10.000 rpm in a slow handpiece for 10 seconds.12 finally, the gutta - percha was compacted vertically using schilder s pluggers (jr instrumental ltda, belo horizonte, brazil). A medium gutta - percha cone 0.06 was adjusted in the same manner as in the previous group . Lateral condensation was performed using a size c finger spreader and fine accessory gutta - percha cones . The gutta - percha excess was removed using heated schilder s pluggers, and, finally, the gutta - percha was compacted vertically using schilder s pluggers . Immediately after filling, postoperative radiographs were taken in the buccolingual direction, and all of them were identically exposed, developed, and fixed . The teeth were cross - sectioned using an isomet precision saw (buhler ltd ., lake bluff, ny, usa) and a diamond disc (125 mm x 0.35 mm x 12.7 mm 330c) at the low speed, placed perpendicular to the main canal at 4 mm, 7 mm, and 10 mm from the apex (1 mm above the point of making the lateral canals). After cross - sectioning, each specimen was immersed in a polyester resin (cebtrofibra, fortaleza, brazil) to make their manipulation simpler (figure 1d). The blocks were polished using specific sandpaper (dp - netot 4050014-struers, ballerup, denmark) for materialographic preparation . The specimens were polished prior to their examination under the stereoscopic lens using a diamond paste of 4 - 1 m roughness (sapuq 40600235, struers) and sandpaper size 1000 . This was done to avoid gutta - percha deformation and to obtain a surface that was free from scratches and deformities, resulting in a highly reflective surface.13 images were obtained (figures 2 and 3) using a nikon coolpix e4.300 pixel digital camera (nikon corp . Korea) connected to a stereoscopic lens (lambda let, hong kong, china) (40x). Radiographic analysis and a filling linear measure (figure 4) using the image tool 3.0 program (university of texas) were performed . For the radiographic analysis, a lateral canal qualified as filled when it appeared to be filled to the external surface of the root ., chicago, ill, usa), and this software indicated the kruskal - wallis test (nonparametric test, samples not normal) to test the null hypothesis that there was no relationship between filling technique and the filling ability of the simulated lateral canals with gutta - percha . Ltda, belo horizonte, brazil) was fitted to working length using a calibrator rule (dentsply maillefer, petrpolis, rj, brazil) that adjusts the tip of the cone to the size 40 k - file . The heat source was activated, and a preheated medium plugger (system b, analytic technology, glendora, ca, usa) was inserted into the root canal to thermoplasticize and compact the gutta - percha at the apical third, 5 mm from the apex . Vertical compaction was performed using a size 2 plugger (jr instrumental ltda, belo horizonte, brazil). The middle and coronal thirds of root canal were backfilled using obtura ii (obtura corporation, penton, missouri, usa). A medium gutta - percha cone 0.06 was adjusted in the same manner as in the previous group . Lateral condensation was performed using a size c finger spreader (dentsply maillefer ballaigues, switzerland) and fine accessory gutta - percha cones . The gutta - percha excess was removed using a heat plugger, and the gutta - percha mass was thermomechanically compacted at the coronal and middle thirds of the root canals using a size 45 gutta - condensor (dentsply maillefer, ballaigues, switzerland) rotated at 10.000 rpm in a slow handpiece for 10 seconds.12 finally, the gutta - percha was compacted vertically using schilder s pluggers (jr instrumental ltda, belo horizonte, brazil). A medium gutta - percha cone 0.06 was adjusted in the same manner as in the previous group . Lateral condensation was performed using a size c finger spreader and fine accessory gutta - percha cones . The gutta - percha excess was removed using heated schilder s pluggers, and, finally, the gutta - percha was compacted vertically using schilder s pluggers . Immediately after filling, postoperative radiographs were taken in the buccolingual direction, and all of them were identically exposed, developed, and fixed . The teeth were cross - sectioned using an isomet precision saw (buhler ltd ., lake bluff, ny, usa) and a diamond disc (125 mm x 0.35 mm x 12.7 mm 330c) at the low speed, placed perpendicular to the main canal at 4 mm, 7 mm, and 10 mm from the apex (1 mm above the point of making the lateral canals). After cross - sectioning, each specimen was immersed in a polyester resin (cebtrofibra, fortaleza, brazil) to make their manipulation simpler (figure 1d). The blocks were polished using specific sandpaper (dp - netot 4050014-struers, ballerup, denmark) for materialographic preparation . The specimens were polished prior to their examination under the stereoscopic lens using a diamond paste of 4 - 1 m roughness (sapuq 40600235, struers) and sandpaper size 1000 . This was done to avoid gutta - percha deformation and to obtain a surface that was free from scratches and deformities, resulting in a highly reflective surface.13 images were obtained (figures 2 and 3) using a nikon coolpix e4.300 pixel digital camera (nikon corp . Korea) connected to a stereoscopic lens (lambda let, hong kong, china) (40x). Radiographic analysis and a filling linear measure (figure 4) using the image tool 3.0 program (university of texas) were performed . For the radiographic analysis, a lateral canal qualified as filled when it appeared to be filled to the external surface of the root . Data were statistically analyzed using spss 12.0 for windows (spss inc ., chicago, ill, usa), and this software indicated the kruskal - wallis test (nonparametric test, samples not normal) to test the null hypothesis that there was no relationship between filling technique and the filling ability of the simulated lateral canals with gutta - percha . The teeth in group 1 (continuous wave of condensation) had the largest number of filled lateral canals in the radiographic analysis, followed by group 2 (thermomechanical technique) and group 3 (lateral condensation) (table 1). The coronal third had a larger number of filled lateral canals than the middle and apical thirds, in the radiographic analysis (table 2). Group 2 had the largest linear measure percentage of simulated lateral canals filled with gutta - percha and sealer (p<.05). No statistical differences were found between group 1 and group 2 when we analyzed the filling with gutta - percha and sealer or just sealer (p>.05). No statistical differences were found between group 1 and group 3 when we analyzed the filling with just sealer (p>.05) (table 3). Groups 1 and 2 had the largest linear measure percentage of lateral canals filled (gutta - percha and sealer) and were statistically different from group 3 (p<.01). The ramifications in the main canal, such as lateral canals, have great clinical importance in endodontic therapy, mainly when associated with lateral lesions . The vertical condensation of the warm gutta - percha technique produces consistently dense, dimensionally stable, three - dimensional root canal fillings . Lateral canals are filled with extraordinary frequency, often with gutta - percha and sometimes with sealer.14 the aim of root canal treatment is to fill the root canal system and isolate microorganisms in inaccessible areas, without access to space and nutrients . Thus, we reestablish the normal condition of the periapical tissue.15 the use of sealers which have good flow rates, among other requisites, and whose composition is based on inert materials, is necessary.16,17 there are several studies dealing with lateral canals . Some of them employed epoxy resin blocks;11,18,19 others simulated lateral canals in natural human teeth.16,20,21 the authors are aware of the limitations of using epoxy blocks versus natural teeth . The surface texture and resin conditions could positively or negatively influence the flow properties of the gutta - percha or sealer . Dentin is an excellent thermal insulator, whereas this property in acrylic substrates is unknown . However, the use of resin allowed us to create an ideal canal shape for filling and the standardized lateral canal.11,19,22 in this study, to reproduce the conditions observed in clinical practice, simulated lateral canals were created having a diameter of approximately 150 m, which is in accordance with the size of lateral canals reported in previous studies.23,24 the lateral canals showed a slightly tapered shape, with a base side to the external root surface . However, the taper does not improve the flow of filling material, because the base of the taper is on the root external surface . X - ray analysis,20,21 one - dimensional observation in epoxy blocks,11 and tooth - clearing techniques22 were used to evaluate the lateral canal fillings . The filling quality of each lateral canal could be better analyzed by cross - sectional comparison within and between each technique.11 in this present study, a cross - section of the root canals was cut through points over the lateral canals . After the specimens were set in epoxy resin and the resin polished, photos were taken at 40x magnification, and the images permitted us to view the presence or absence of gutta - percha and sealer in each lateral canal . In this way, other kinds of analyses could be performed . Several studies have reported endodontic success after filling of lateral canals and different filling techniques were proposed to achieve better obturation of these canals.11,14 although some studies cast doubt on the idea that the type of canal filling technique has a great effect on the number of lateral canals filled,9,20 other studies have demonstrated that vertical compaction of warm gutta - percha increases the capacity of lateral canal filling.14,21 thermoplasticized techniques and cold techniques were compared in 360 simulated lateral canals . According to the results given by x - ray analysis, the thermoplasticized techniques filled more lateral canals.20 in particular, the thermoplasticized techniques allow the high compaction forces to be directed towards the gutta - percha within the accessory canals, resulting in a complete three - dimensional filling of ramifications of the middle and coronal regions.22 in this present study, the thermoplasticized techniques, either heat source or mechanical, filled more lateral canals than the lateral condensation technique, and the apical third was less filled than the other thirds . Groups 1 and 2 had 91.26% and 95.93% of the extension of lateral canals filled with gutta - percha and sealer, respectively . Group 2 had more gutta - percha in the lateral canals than did the other two groups . This fact may have been caused by the lateral force of the gutta - condenser rotation . The literature have reported that the warm gutta - percha technique promotes better filling quality and results in virtually no gaps, very low amounts of sealer on the root surface, and greater adaptation to dentinal walls, unlike the lateral condensation technique.15 in this study, when thermoplasticized gutta - percha techniques were used, a greater frequency of filled lateral canals was noted compared with lateral compaction of gutta - percha . Thermoplasticized gutta - percha techniques were better than lateral condensation technique to fill simulated lateral canals . Thermoplasticized gutta - percha filling techniques are better for filling lateral canal with gutta - percha and sealer or with just sealer than lateral condensation . However, the present study was performed in vitro, and it is not possible to establish a direct correlation with clinical findings . Moreover, it should be noted that, although the results suggest the superiority of one technique over another, there is no scientific evidence proven through clinical studies.
Human immunodeficiency virus (hiv) and acquired immune deficiency syndrome (aids) are associated with multiple endocrine abnormalities . Several investigators have studied the metabolic derangements and reported the functional abnormalities with specific endocrine glands . There is however scant literature regarding histopathology of the thyroid and parathyroid glands in patients with hiv and aids, particularly in african americans (aa). Nevertheless, extensive literature is available on serum biochemical thyroid functions and calcium fluctuations in these patients . Euthyroid sick syndrome, hypothyroidism, hypocalcemia, impaired parathyroid hormone secretion, and vitamin d deficiency appear to be common among the adult hiv - infected patients [35]. We postulated that the thyroid and parathyroid glands would be involved in aids patients as evidenced by functional abnormalities and derangements seen in these patients . Our study is the first retrospective report to describe the histopathology of the thyroid and parathyroid glands in hiv - infected aa patients in an inner city hospital in the united states . A retrospective review of histopathology findings of the thyroid and parathyroid glands at autopsy was conducted at a tertiary care teaching hospital during the year 1980 through 2007 . One hundred and two hiv - infected patients who died after admission to howard university hospital were identified . The study was reviewed and approved by the institutional review board . At the time of autopsy, two transverse sections of the thyroid gland were routinely performed . If pathological lesions were noted, multiple sections were performed . Other stains included periodic acid schiff, grocott's methenamine silver and ziehl - neelsen stain as necessary . The starting time of the study coincided with the introduction of highly active antiretroviral treatment (haart); hence, no many patients were on haart . Incomplete adherence to the prescribed regimen was common, as self - reported by patients as well as evidenced by the number of hiv associated opportunistic infections these patients harbored . We compared the histopathological findings to a control - group which included patients without hiv infection (31 females and 44 males) who died at the hospital from other causes . There were 71 males (70%) and 31 females (30%) with an average age of 38 (range: 2071 years). The mean time from hiv diagnosis to death was 53 months (range: 1144 months). Most of the patients had aids, as suggested by the number of opportunistic infections they had (% patients had aids defining illnesses). Cd4 lymphocyte count was documented in 20.5% of patients with a median cd4 count of 50 cells/l . Thirty - four (33%) of patients were from the pre - haart period (19801990). Our series included 40 patients with intravenous drug abuse (39.2%), 23 (22.5%) with heterosexual risk, 23 (22.5%) with homosexual or bisexual risk, and 56 (55%) with undocumented sexual preference . It is to be noted that these high risk behavior patterns were not mutually exclusive . A control group for thyroid histopathologic examination was obtained in 75 non - hiv - infected patients . Comparing the histological findings of cases and controls, we found similar involvement of the thyroid, with greater prevalence of parathyroid hyperplasia in hiv patients . The thyroid gland weighed between 3 to 40 g. weights of parathyroid glands were not available . Thirteen patients (7 males and 6 females) had abnormal histopathology of the thyroid gland . The mean time from hiv diagnosis to death was 65.8 months ranging from 6 to 132 months . Only 4 of 13 patients had a cd4 count available with a mean 64, range of 0200 . One patient in this group was receiving pneumocystis jiroveci prophylaxis and 4 patients were on haart . The mean body mass index (bmi) was 25 kg / m with a range of 1540 kg / m . The mean weight of the thyroid gland was 21.4 grams with a range of 3 to 32 grams . The gross examination of the thyroid gland was unremarkable in 100 patients (98%). One patient had a macroscopically nodular thyroid gland while another patient had an atrophic thyroid gland . The latter patient had both macroscopic (atrophy) and microscopic abnormalities (fibrosis) (table 1). In the control group, the macroscopic histopathologic analysis of the thyroid revealed 68 patients (90.6%) having a normal thyroid macroscopic examination (table 1). Interstitial fibrosis (figure 1) was the most common histological finding identified in thyroid gland sections (4.9%), followed by thyroid hyperplasia (1.9%). One case of colloid goiter and one case of thyroid adenoma were also identified at autopsy (table 3). Infections of the thyroid gland included cytomegalovirus (1 case), mycobacterium tuberculosis (1 case), and cryptococcus (1 case), (figures 2 and 3). Furthermore, the most common systemic opportunistic infection in 13 of our patients with thyroid abnormalities was mycobacterium avium complex infection (mac) (38.4%) followed by candida albicans (figures 4 and 5). In the remaining hiv - infected patients without thyroid abnormalities, the most frequent opportunistic infection was pneumocystis jiroveci (32.5%), followed equally by candida and cytomegalovirus (19.1%). Parathyroid hyperplasia was by far the most common histological finding accounting for 22.5% of cases followed by cytomegalovirus (cmv) infection of the parathyroid (2.9%) and parathyroid hyperplasia was diagnosed if at least two of all four parathyroid glands were hyperplastic . Fatty infiltration (1.9%) and serous atrophy (1.9%) were also identified in the parathyroid glands (table 4) (see figures 6 and 7). Most of these patients studied died of septic shock or respiratory failure (data not shown). Review of patient's data showed that abnormal pathological findings were found entirely in patients with ongoing illicit drug use . The histological findings in control patients revealed cytological appearances consistent with benign thyroid nodular disease in 8% of control patients, interstitial fibrosis in 2.6%, lymphocytic thyroiditis in 2.6%, cryptococcal infection in 1.3%, papillary carcinoma in 1.3%, and mycobacterial tuberculosis in 1.3% of the control specimens (table 3). The histological appearance of parathyroid glands from the control group did differ from the hiv group with 72 control patients (96%) showing normal histological appearance of the parathyroids as opposed 69 hiv - infected patients (67.6%) (table 2). Patients with aids have increased prevalence of nonthyroidal illness, hypothyroidism, and abnormal serum parathyroid hormone (pth) and serum calcium levels [4, 5]. These alterations in thyroid hormones and calcium homeostasis are rarely the result of a direct infection or infiltration of the thyroid and parathyroid glands . Although subclinical hypothyroidism has been recognized as more prevalent among hiv - infected individuals, it does not appear to have an autoimmune basis . Graves's disease subsequent to immune restoration due to haart has been described and unlike the common infection - related immune reconstitution syndromes, it is usually diagnosed 1236 months after haart initiation . Two studies from south america have described pathological changes in the thyroid gland in aids patients [8, 9]. However, no investigator has reported the histopathology of parathyroid glands in human immunodeficiency virus (hiv) patients . This study represents the first detailed report of thyroid and parathyroid gland abnormalities in a hiv - infected african - american population in the united states . Ethnicity - related differences in organ systems involvement in hiv patients have been described previously by morgello et al . With cachexia, renal, cardiac and splenic involvement more frequent in blacks than in whites and/or hispanic individuals . Additionally, mycobacterium avium - intracellulare (mai) infection is also more commonly seen in blacks than in whites and/or hispanic individuals . Our findings are strikingly different from what have been published so far in terms of the frequency of thyroid involvement in hiv . Mycobacterium tuberculosis infection of the thyroid gland was found in 23% of patients and cytomegalovirus (cmv) in 17% . Neoplastic involvement of the thyroid was also higher in frequency with kaposi sarcoma (2%) and occult papillary carcinoma (4%) seen in patients . Studied forty - seven thyroids obtained at autopsy from 38 men and 9 women with aids in brazil . In contrast to our results, they identified greater frequency of infectious pathogens (14 cases, 29.7%) with five cases of mycobacterial infection (10.6%), four cases of histoplasmosis and cryptococcosis, and finally one case of paracoccidioidomycosis . Their results were concordant with baslio - de - oliveira in regard to mycobacterium infection being the most frequently detected agent . This may be due to the higher prevalence of mycobacteria in aids patients in brazil [11, 12]. In postmortem examinations of these patients, thyroid pathology was common affecting 29 patients (61.3%), with nonspecific focal chronic inflammation affecting 14 cases (48.2%), colloid goiter in 5 cases (17.2%), and lipomatosis in 4 cases (13.7%). Lipomatosis was associated with atrophy (1 case), hyperplastic nodule (1 case), and histoplasmosis (1 case). In our study, the frequency of infectious etiology affecting the thyroid gland was limited to 2.9% (3 cases).there was only one case of cytomegalovirus (cmv), cryptococcus, and tuberculosis . All our cases inclined to have occurred in the context of a widely 5 disseminated disease . Several cases have been previously reported also as part of multiple organ involvement either antemortem or at autopsy [1317]. In our series, some patients presented with multiple coexisting opportunistic infections . About 45% of patients in the hiv group with thyroid pathology had more than 2 opportunistic infections as opposed to 35% in the subgroup of hiv patients with normal thyroid histopathology (table 5). Based on the limited number of patients, we cannot postulate a possible association between opportunistic infections and the occurrence of thyroid abnormalities . While in previous case reports, pneumocystis jiroveci has been a prominent cause of thyroiditis in hiv patients [1821], particularly in patients on aerosolized pentamidine [22, 23], we did not identify this microorganism in any of our patients . This may be due to the fact that many patients presented to our hospital with symptoms suggestive of pneumocystis jiroveci pneumonia and were treated promptly with trimethoprim - sulfamethoxazole leading to absence of histological evidence of this microorganism at autopsy . This observation is consistent with other studies showing that after curing patients of their pneumocystis infections, biopsy specimens usually lack evidence of residual disease [24, 25]. Our most common finding was interstitial fibrosis seen in 5 of the 12 microscopic cases . Have linked thyroid fibrosis to transforming growth factor beta (tgf - beta) in which follicular cell necrosis occurs first followed by thyroid fibrosis in the setting of selenium deficiency . Similarly, interstitial fibrosis of the thyroid gland in our series could represent the histologic sequelae of previous inflammatory or infectious assaults coupled with an impaired tissue repair due to the underlying immunosuppression . In addition, human immunodeficiency virus infection itself is associated with increased levels of transforming growth factor beta (tgf - beta). Of note, we identified two cases of thyroid hyperplasia which has been described as a normal response to alterations in the feedback mechanism of thyrotropin - releasing hormone and thyroid - stimulating hormone . Additionally, it has been shown that hiv-1-infected inflammatory cells may release a mitogen protein (tat) which enhances the production of growth factors including fibroblast growth factor (fgf-1 and fgf-2) and transforming growth factor - beta . Kaposi sarcoma of the thyroid is uncommon, described only in the context of a widespread metastasis . Little is known about the relative contribution of illicit drugs use to the thyroid histopathology in hiv - infected populations . The histologic appearance of parathyroid hyperplasia was hypercellularity with heterogenous cell proliferation but predominantly chief cells associated with reduced stromal fat and involving more than one gland . This finding could be reflective of the secondary hyperparathyroidism resulting from the high prevalence of vitamin d deficiency in african american in general and also specifically hiv - infected individuals [35, 36], although we do not have vitamin d levels in any of these patients . Both parathyroid and nodular oncocytic hyperplasia have been described as a feature of secondary hyperparathyroidism . In the hiv population in particular, this process can also result from decreased serum calcium secondary to impairment in renal function, nutritional status, and chronic malabsorption . Of note, a decrease of parathyroid hormone (pth) level has also been previously reported in human immunodeficiency virus (hiv)-infected patients . The mechanism might be related to antibodies against parathyroid cells . Using anti - leu3a, a monoclonal antibody recognizing cd4, hiv - positive patients have been found to express cd4 molecule at the surface of parathyroid gland cells, indicating the possibility of either functional inhibition by anti - cd4 antibodies or direct infection by hiv . Therefore, the question of whether parathyroid hyperplasia could be related to possible micronutrient deficiency such as iodine deficiency remains to be established in hiv - infected subjects . Further research is needed to elucidate the role of micronutrient deficiencies on parathyroid pathology in hiv - infected subjects . First, our findings stem from a retrospective review of general autopsies in hiv - infected african american patients; hence, the small number of histologic sections might have influenced the microscopic findings recorded . Larger studies focusing on thyroid and parathyroid are required to establish the prevalence of our findings . Second, we were unable to analyze the thyroid function tests and the autoimmune status of our sample . One could argue that interstitial fibrosis is fairly non - specific and further prospective studies correlating histopathological findings with thyroid serologies for a better assessment of thyroid pathology in hiv african american population are needed . We conclude that thyroid and parathyroid abnormalities are uncommon findings in the hiv - infected african american population . The most common characteristics of histopathology seen in the thyroid and parathyroid glands in these patients include interstitial fibrosis and parathyroid hyperplasia, respectively.
Castleman disease (cd) is a rare lymphoproliferative disease, which is histopathologically classified into hyaline vascular type, plasma cell type and mixed type . Although the cause of this disorder has not been definitely established, chronic low - grade inflammation, immunodeficiency state and autoimmunity are the proposed pathogenetic mechanisms . Several autoimmune diseases have been associated with cd, but no association with hashimoto thyroiditis has been defined . The standard for staging of the disease is contrast - enhanced computed tomography (ct). There is insufficient data in the literature about the role of [f]fluorodeoxyglucose (fdg)-positron emission tomography (pet)/ct in staging and evaluating response to treatment . This case report discusses a patient with unicentric mixed type cd associated with hashimoto thyroiditis, for whom staging and evaluation of response to therapy was done with fdg - pet / ct . A 62-year - old woman was admitted to the hospital with atypical chest pain . The physical examination and electrocardiography were normal . The ct scan of the chest revealed multiple mediastinal lymphadenopathies (laps) extending from the thyroid level to the aortic root and surrounding the major vascular structures . The pet / ct study showed the presence increased fdg uptake in the perivascular structures of the anterior mediastinum (fig . Also, a diffuse fdg uptake was determined in both lobes of the thyroid gland . As a result, figure 1fdg - pet / ct images at diagnosis: (a) selected coronal pet slice, (b) corresponding ct slice, (c) maximum intensity projection (mip) image of pet data . Fdg - pet / ct images after treatment: (d) coronal pet slice, (e) corresponding ct slice, (f) mip image of pet data . Fdg - pet / ct images at diagnosis: (a) selected coronal pet slice, (b) corresponding ct slice, (c) maximum intensity projection (mip) image of pet data . Fdg - pet / ct images after treatment: (d) coronal pet slice, (e) corresponding ct slice, (f) mip image of pet data . Histological analysis revealed hyaline vascular type cd in one lymph node and plasma cell type cd in 3 lymph nodes . 2). Figure 2(a) the plasma cells and some blood vessels stained with haematoxylin eosin (he 20); (b) plasma cells seen in more detail with microscopic zoom (he 40); (c) plasma cells are positive with cd79a immunohistochemically (cd79a 20); (d) hyaline vascular variant type, plasma cells are seen in an eosinophilic hyalinized area (he 40). (a) the plasma cells and some blood vessels stained with haematoxylin eosin (he 20); (b) plasma cells seen in more detail with microscopic zoom (he 40); (c) plasma cells are positive with cd79a immunohistochemically (cd79a 20); (d) hyaline vascular variant type, plasma cells are seen in an eosinophilic hyalinized area (he 40). Laboratory evaluation showed anaemia of chronic disease (hb 9.3 g / dl), elevated erythrocyte sedimentation rate (65 mm / h), mild elevation of serum igg level (17.1 g / l, normal limits 716 g / l), and hypothyroidism (thyroid stimulating hormone 24.98 iu / ml). Bone marrow aspiration and biopsy showed a normocellular bone marrow with no increase in plasma cell count . Antithyroglobulin antibody level was> 4000 iu / ml (normal range 0115 iu / ml) and her antithyroid peroxidase antibody level was 331.7 iu / ml (normal range 034 iu / ml). The patient was diagnosed as hashimoto thyroiditis and levothyroxine 0.1 mg was initiated . Further evaluation for autoimmune diseases showed no disorders other than hashimoto thyroiditis . Due to the close relationship of the laps to the major vascular structures, complete surgical resection was not feasible . As the patient had a medical history of asthma and mild cardiac dysfunction, mediastinal radiotherapy was not considered to avoid cardiac and pulmonary toxicity . The patient received 8 cycles of vincristine (2 mg, day 1) and prednisolone (100 mg, days 15), every 2 weeks . After 8 cycles of chemotherapy, the laps had regressed 30% in diameter on chest ct . F). During the 42-month follow - up, no clinical and radiological signs suggestive of recurrence have been seen . Cd is a polyclonal lymphoproliferative disease, characterized by lymph node enlargement with distinctive histological and clinical features . It has been divided clinically into unicentric and multicentric types, and histopathologically into hyaline vascular type, plasma cell type and mixed type the hyaline vascular variant is the most common, comprising 90% of cases and is characterized by a benign clinical course . In contrast, the plasma cell variant accounts for 9% of cases and tends to be clinically aggressive . Malignant transformation and systemic findings such as fever and anaemia are rare in this variant of cd . The plasma cell variant is generally multicentric and in contrast to the hyaline vascular type, systemic findings are frequently encountered, as well as a high risk of malignant transformation . Mixed variant cd is mostly multicentric, however, as in our patient, some patients with unicentric cd reveal features of both hyaline vascular type and plasma cell type . Although the cause of cd is unknown; chronic low - grade inflammation, immunodeficiency state and autoimmunity have been proposed as likely pathogenic mechanisms numerous autoimmune diseases are associated with cd including sjgren syndrome and rheumatoid arthritis . Dysregulated interleukin-6 production may play a key role in the pathogenesis of cd . In our case, it is not clear if autoimmunity is the underlying cause of cd, as it has been suggested that cd can alter the immune system and might lead to development of such an autoimmune disease . So, the autoimmune diseases can be the cause or the result of cd . Although they may share a common pathogenic pathway related to immune dysregulation, the possibility of a coincidental association must also be raised, especially when the high prevalence of hashimoto thyroiditis is considered . The standard modality for the evaluation of disease extent is contrast - enhanced ct, which shows enhancement due to hypervascularity of the lesions . Although the value of fdg - pet / ct in staging has been reported in a few cases, currently the role of fdg - pet / ct in diagnosis and in staging is still controversial . In a case presented by pelosi et al ., it was stated that pet / ct could be considered in staging or restaging the disease and evaluation of response to the treatment . In our case, fdg - pet / ct correctly staged cd at diagnosis and was superior to ct scan in response evaluation . Although ct evaluation demonstrated a partial radiological response, the pet / ct revealed complete metabolic response . Thus, fdg - pet / ct seems to be useful for both assessment of the extent of the disease and response evaluation . The treatment of cd depends on the extent and in some circumstances on the histological subtype . Radiotherapy is the best choice if complete resection is not possible; especially for the plasma cell variant . In contrast to the unicentric variant, the multicentric variant necessitates aggressive systemic therapy due to its high malignant transformation potential and poor prognosis . The systemic treatment options are immunosuppression, cytoreductive agents, immunomodulators (steroids, interferon - alpha, thalidomide) and monoclonal antibodies (anti - interleukin 6, rituximab). In this case, we achieved a durable complete response with chemotherapy in the absence of local therapy such as surgery or radiotherapy . This case report shows that fdg - pet / ct can have an important role in the staging and evaluation of treatment response in cd . We suggest that due to the rapid progression and high metabolic activity of the mixed type variant and the plasma cell variant, assessment of treatment response can be done with pet - ct . This case is also unique as there no case of cd in association with hashimoto thyroiditis has been reported previously.
The prevalence of primary open angle glaucoma (poag) is estimated to be between 1.1% and 3% in western populations over the age of 40 years [1, 2]. The estimated number of poag patients in israel is 60,000 . A large survey study including 10,000 israeli participants demonstrated a similar distribution of the disease among various israeli ethnicities, including arabs . In the literature, compliance is reported to vary from as low as 5% to as high as 80% . Moreover, similar studies and information in the specific israeli - arab population are still lacking . The aim of this study is to estimate the degree, components, and determinants of noncompliance among poag arab patients in israel and to verify associated factors for noncompliance . Glaucoma is insidious and disabling; the degree of interference with vision varies from imperceptible changes to complete blindness . Several authors have indicated that noncompliance is an important factor in blindness resulting from glaucoma [69]. Poag patients attending out - patient eye clinics at the 3 cities, tira, taibi, and qalanswa in israel, were invited to join the study . Subjects were included if they were willing to participate, signed an informed consent, and fulfilled all of the following conditions: were a resident of one of the 3 cities (tira, taibi, or qalnsawa) and from an arab origin ethnicity;were diagnosed by an expert ophthalmologist as having poag for at least 3 months prior to the interview; the diagnosis was based on glaucomatous cupping of the optic disc and a glaucomatous visual field loss, in one or both eyes, regardless of the intraocular pressure;were prescribed medications for glaucoma, in the past 3 months;had no mental or physical condition rendering them unable to participate in the study.exclusion criteria: evidence of angle closure glaucoma (acg). Acg represents a group of ocular diseases with various causes that have a unique behavior and different treatment modalities (such as laser iridotomy); cataract surgery within 30 days prior to the study . Elevated intraocular pressure, following cataract surgery, occasionally requires a limited course of therapy to lower intraocular pressure perioperatively . Were a resident of one of the 3 cities (tira, taibi, or qalnsawa) and from an arab origin ethnicity; were diagnosed by an expert ophthalmologist as having poag for at least 3 months prior to the interview; the diagnosis was based on glaucomatous cupping of the optic disc and a glaucomatous visual field loss, in one or both eyes, regardless of the intraocular pressure; were prescribed medications for glaucoma, in the past 3 months; had no mental or physical condition rendering them unable to participate in the study . Acg represents a group of ocular diseases with various causes that have a unique behavior and different treatment modalities (such as laser iridotomy); cataract surgery within 30 days prior to the study . Elevated intraocular pressure, following cataract surgery, occasionally requires a limited course of therapy to lower intraocular pressure perioperatively . A closed questionnaire, filled solely by medical doctors, was used to collect data during the interview . The first section included general data of the patient, such as age and gender . The participants were asked three short questions: a list of prescribed glaucoma medications;the frequency of daily usage;the frequency of weekly and monthly usage . A list of prescribed glaucoma medications; the frequency of daily usage; the frequency of weekly and monthly usage . Data retrieved, from both the out - patient clinic records and the questionnaires, were used to define compliance and noncompliance . Noncompliance was defined as missing at least one drop of medication per week and (or) the inability to accurately describe one's own medication regimen . Subjects whom were considered as noncompliant were asked to choose one of the eight listed reasons to explain nonadherence (see the appendix). The noncompliance - related questions covered information regarding how to use eye drops, the importance of treatment and consequences following non - adherence, intolerance, and patient - physician interaction . Responses were grouped and analyzed into 5 categories by interviewing doctors (see the appendix). Multiple logistic regression analysis was performed to predict correlation between age and number of anti - iop drugs . In addition, chi - square tests were carried out in order to examine the relationships between gender and compliance and number of anti - iop drugs . Mean age and sd among compliance patients group and noncompliance patients group were similar (table 1). We found a positive statistical significance in the correlation between age and number of prescribed drugs (p <0.05). Logistic regression analysis revealed that age cannot predict compliance (p> 0.05). Interestingly, the compliance was significantly higher among the two groups at either end of the curve, that is, younger than 50 years (77%) and older than 81 years (63%) (p <0.05). Though patients that were prescribed 3 drugs were slightly more noncompliant, the difference is statistically not significant and compliance rates were not influenced by gender or by number of anti - iop drugs (table 2). Lack of knowledge (32%), misunderstanding (25.5%), and denial (15.5%) were the most common reasons indicated . In addition, 14% of the participants mentioned that they could not tolerate the drops, while 13% of the patients could not point to specific reasons for their noncompliance . The aim of this study was to find the noncompliance rates among coag israeli arab patients and to try to define the reason for their noncompliance . The main findings of our survey are the relatively high noncompliance rates among coag israeli arab patients . A pleasing bias may occur, in which patients may tend to orally report better compliance . Furthermore, objectively measuring the effect of therapy, that is, the iop, was not performed . However, in addition to the gathered data from the questionnaires, out - patient clinic records were retrieved and used together to define noncompliant patients, thus minimizing the aforementioned bias . Glaucoma patients who were not patients at outpatient eye clinics at tira, taibi, and qalnsawa cities in israel are not represented in this study . These unreported patients may be receiving treatment at private clinics or may have ceased to participate in their personal treatment plans . Therefore, our study results represent only israeli - arab glaucoma patients treated in the outpatient clinics and may not represent all those suffering from glaucoma among the israeli - arab population . Furthermore, the study relies on patients' responses and involves health - care providers . Better ways to measure disease progression compliance with medication than cross - sectional studies exist, implying that longitudinal studies are needed in the future to evaluate different levels of noncompliance . Results of this study demonstrate that over 50% of the israeli - arab glaucoma patients are not compliant to their treatment plans . Lack of knowledge and misunderstanding are the major reasons given by patients explaining their non - adherence . The reported noncompliance rates in western populations and in the rest of israel vary from 27.4% to 42% according to different studies . Various study designs and nonuniform compliance definitions the relatively high levels (50%) of noncompliance in the primarily israeli - arab population are consistent with many reported population - based studies in certain ethnic groups [12, 13]. For example, african americans are not only more likely to develop poag compared to white americans, but also less compliant in their treatment plans . Similarly, a recent study reported an even higher nonadherence rate among oman glaucoma arab patients (up to 75.2%). Purportedly, a higher rate of noncompliance among females (52.7% compared to males 47.2%) was observed in our survey . Yet, the differences are statistically insignificant, implying that gender per se is not associated with increased noncompliance [14, 15]. Figure 1 describes a positive association between age and number of anti - iop drugs . Although overall noncompliance rates increased from 28.5% with once daily drug to 38% with 3x daily, this increment was statistically insignificant (table 2). In addition, the frequency of instillation per day did not affect compliance significantly, although studies have shown that a single daily drug dose or combination is associated with better compliance rates [15, 16]. Granstrom and norell have clearly shown that 4x daily regimen of pilocarpine was unrelated to low compliance . However, noncompliance, in this study, is related to the patient's experience of side effects and/or negative attitudes to the treatment . In our study, significantly higher rates of compliance were found among the two extreme age subgroups, that is, <50 years old and> 80 years old (see figure 2), 63% and 77%, respectively . Younger glaucoma patients are found to have a tendency to follow treatment regimens strictly probably due to proper perception of their disease and better instillation compared to older individuals . As for the elderly, gurwitz et al . Found that elderly glaucoma patients started on multiple glaucoma medication were more adherent than those started on a single agent . Therefore, in our older glaucoma group, a high probability of multiple medications might explain the high compliance rates . However, several studies clearly indicate higher prevalence rates of noncompliance among elderly, mainly due to improper installation of eye drops as well as other age - related medical illnesses, such as dementia and arthritis . Clearly, a major determinant of compliance with medical treatment in any medical illness is the patient's awareness and understanding of his disease . Poag usually produces few external signs, making the severity of the disease more difficult to perceive and possibly causing later complications, particularly because the only symptoms may be the side effects of the medical treatment prior to later more serious complications such as visual field loss . Lack of knowledge and misunderstanding were the main factors cited for the> 57% noncompliance rates in our patients . The association between compliance and awareness of the disease, its severity, treatment, and its late complications has been previously noted [9, 14, 20]. Furthermore, patients with higher levels of formal education are more accurate in describing their medication regimen . In addition, better knowledge about glaucoma is associated positively with better compliance rates . In a recent study in oman, the knowledge about glaucoma was reported to be good in 23.8% of patients, who demonstrated higher compliance rates . Reported that over a third of glaucoma patients failed to use their ophthalmic medications properly, mainly due to lack of knowledge . Many patients are in denial about their health problems, especially in asymptomatic diseases such as glaucoma . Not surprisingly, denial and intolerance comprised 30% of the noncompliant patients . In addition, the patient - physician interaction remains a cornerstone in improving compliance rates due to intolerance [7, 11, 15]. The physician should tailor the medication to the patient's lifestyle after careful investigation of the patient's routines such as sleeping, eating, and daily activity patterns [11, 14, 15]. Additionally, the physician must be aware of possible side effects and keenly seek them out, while always discussing various treatment options . Noncompliance was found to be relatively common among israeli - arab coag patients in both sexes . The overall noncompliance rate was 50% and was not influenced by gender or by number of anti - iop drugs . Compliance was significantly higher among patients younger than 50 years (77%) and older than 81 years (63%). The most common reasons indicated for noncompliance were lack of knowledge, misunderstanding, and denial . Educating patients about their disease and its complications, using guiding brochures and dvds, and improving the patient - physician relation by personalizing the treatment can plausibly improve compliance rates . Furthermore, patients' needs and knowledge should be taken into consideration in order to improve patients' compliances.
The retrorectal space is an uncommon area where tumors occur and these include primary tumors of neurogenic, osteogenic, and congenital origin; in addition to metastatic and inflammatory processes . Congenital lesions include chordomas (remnants of notochord), teratomas, anterior sacral meningoceles, and developmental cysts (dermoid, epidermoid, enteric duplication, and tailgut cysts (tgcs)). Tgcs, also known as retrorectal cystic hamartomas, are a rare congenital lesion thought to arise from the remnants of the embryonic postanal gut . Hjermstad and helwig were the first to publish their findings in 1988, and since then there have been no large case series reported . From review of the literature done by killingsworth and gadacz (keyword = tailgut cyst or retrorectal cystic hamartoma, limits = english), there have been 43 cases with confirmed diagnosis of tgc since their report . A 15-year - old girl presented with the complaints of lower abdominal pain and constipation occasionally . However, on per rectal examination, there was a mass bulging from the posterior rectal wall, firm, and non - tender, with regular surface and smooth mobile rectal mucosa over it . An ultrasonogram (abdominal) revealed a large cystic lesion present in the left lower abdomen and the left ovary could not be seen separately . The patient then underwent a contrast - enhanced computed tomography (cect) of the abdomen and pelvis which revealed a well - defined 12 13 9 cm multiseptated lesion in the presacral space which was pushing the rectum laterally and urinary bladder superiorly and abutting the sacrum and coccyx posteriorly [figure 1a and b]. The lesion was showing peripheral and septal calcification, few hyperdense nonehancing areas and few ossified fragments within it . A provisional diagnosis of mature cystic teratoma was made and the patient underwent exploratory laparotomy wherein a large tubular tense cystic mass resembling fluid - filled intestinal loop filled with thick mucoid material was present in the presacral space [figure 2]. The two ends of the tube were merging at the coccyx . The mass was displacing the sigmoid colon and rectum laterally and urinary bladder anteriorly . En masse removal was done . Cect abdomen showing multiseptated pre sacral mass compressing the rectum and displacing bladder superiorly intraoperative picture showing a tubular fluid - filled structure displacing the bowel loops the patient had an uneventful postoperative recovery . The histopathological examination revealed it to be a retrorectal cystic hamartoma with areas of intestinal (large and small) and gastric epithelium . A solitary solid area within it had intestinal lining with area of squamous epithelial nests, haphazardly arranged muscle bundles, nerve bundles, and serous acini with few cystic spaces [figure 3a c]. Histopathological image showing (a) gastric mucosa, (b) ectopic gastrointestinal gland and (c) ectopic pancreatic epithelium the retrorectal space is a potential space developed when a mass displaces the rectum anteriorly . The pelvic peritoneal reflection forms the superior border, and the levator ani and coccygeus muscles form the inferior border . The differential diagnosis of masses within this space is broad and includes primary tumors of neurogenic, osteogenic, and congenital origin; in addition to metastatic and inflammatory processes . Congenital lesions include chordomas, teratomas, anterior sacral meningoceles, and developmental cysts (dermoid, epidermoid, enteric duplication, and tgcs). Excluding inflammatory lesions, developmental cysts are the most common masses in the retrorectal space . . Only one case of a retrorectal cystic hamartoma occurred in a 2-year - old child and very few cases have been reported in teen aged girls, as in our case . The differential diagnosis for a retrorectal mass can be narrowed using a combination of diagnostic tools to reach a preoperative diagnosis of a developmental cyst . Due to their location, almost all retrorectal tumors will be palpable on rectal examination, and developmental cysts will manifest as extrinsic masses . Ct and magnetic resonance imaging (mri) are useful imaging modalities that help in making a preoperative diagnosis . However, the definitive diagnosis and treatment is through complete surgical excision and pathological examination of the specimen . Preoperative biopsy should not be attempted (unless the mass is surgically unresectable at presentation) due to risk of spreading dysplastic cells through weakened cyst walls . In addition, tissue obtained from biopsy is often not extensive enough to show all the histology features necessary for diagnosis . Dermoid and epidermoid cysts are both lined with stratified squamous epithelium; however, only dermoid cysts contain dermal appendages (hair follicles, sweat glands, and tooth buds). Epidermoid cysts are formed from inclusion of epidermal elements at the time of neural groove closure in the meninges . Rectal duplication cysts are lined by typical gastrointestinal epithelium (often with crypts, villi, and glands) and are surrounded by two well - formed layers of smooth muscle with nerve plexuses . Tgcs, or retrorectal cystic hamartomas, are predominantly multicystic and can contain a variety of epithelia between cysts or even within the same cyst . Epithelial types include stratified squamous, transitional, mucinous or ciliated columnar, and cuboidal mucus secreting . In contrast to enteric duplication cysts, tgcs have disorganized smooth muscle fibers within the cyst wall and do not contain neural plexus . Retrorectal hamartoma or tgc should be considered as a possible differential in any case of perirectal cyst, irrespective of age and gender.
In 1983, shafer introduced the term ameloblastic carcinoma to describe ameloblastomas in which there had been histologic malignant transformation . Cases of ameloblastic carcinomas are rare and limited details on prevalence, incidence or relative frequency are presently available . Compared to the metastasizing, malignant ameloblastoma; however, the ameloblastic carcinomas seem to be more common (2:1). Ameloblastic carcinoma occurs in a wide range of age groups, but the mean age of 30.1 years is in agreement with that reported for ameloblastomas . Involvement of the maxilla by ameloblastic carcinoma seems to be less frequent than that of the mandible . The most common sign described has been swelling; although, others include associated pain, rapid growth, trismus and dysphonia . Carcinomas derived from ameloblastomas have been designated by a variety of terms, including malignant ameloblastoma, ameloblastic carcinoma, metastatic ameloblastoma, and primary intra - alveolar epidermoid carcinoma . In 1971, the world health organization, published its classification of odontogenic carcinomas recognizing the following subtypes: malignant ameloblastomaprimary intra - osseous carcinomaother carcinomas arising from odontogenic epithelium, including those arising from odontogenic cysts . Malignant ameloblastoma primary intra - osseous carcinoma other carcinomas arising from odontogenic epithelium, including those arising from odontogenic cysts . In this classification, malignant ameloblastoma refers to a neoplasm in which typical histologic features of ameloblastoma are seen in the primary tumor located in the jaw as well as in any associated metastatic deposits . In 1984, slootweg and mller, further emphasized that ameloblastomas may exhibit malignant features other than metastasis and suggested a modified classification system for malignant tumors with features of ameloblastoma, based on characteristics of malignancy: type 1: primary intraosseuos carcinoma ex odontogenic cysttype 2: (a) malignant ameloblastoma (b) ameloblastic carcinoma, arising de novo, ex ameloblastoma or ex odontogenic cysttype 3:primary intraosseous carcinoma arising de novo (a) non - keratinizing (b) keratinizing . Type 1: primary intraosseuos carcinoma ex odontogenic cyst type 2: (a) malignant ameloblastoma (b) ameloblastic carcinoma, arising de novo, ex ameloblastoma or ex odontogenic cyst type 3:primary intraosseous carcinoma arising de novo (a) non - keratinizing (b) keratinizing . The treatment of and prognosis for ameloblastic carcinoma is unclear in the literature due to the rarity of this tumor and the lack of well - documented patients . Surgical excision, with or without adjuvant radiotherapy, seems to be required for local control . A 44-year - old female patient presented to the outpatient department with a chief complaint of swelling in the lower jaw since 6 months . She had no contributing medical history . On extra oral examination a large well - defined swelling was noticed in the mandibular anterior region crossing the midline causing facial asymmetry [figure 1a]. (a) large well - defined swelling involving the mandibular anterior region crossing the midline causing facial asymmetry, (b) large swelling extending completely into the floor of the mouth and completely obliterating the lingual and buccal vestibules the swelling extended below the inferior border of the mandible and the skin over the swelling was stretched and smooth . Intraoral examination showed a large swelling, which extended completely into the floor of the mouth and completely obliterating the lingual and buccal vestibules mediolaterally [figure 1b]. There was no surface discharge present, the mucosa over the swelling was normal and its color was same as that of the normal tissue . The swelling was bony hard, non - tender, and immobile . None of the teeth present showed mobility . Computed tomography showed a large multilocular osteolytic lesion extending from 37 to 47 region crossing the midline, destruction of both cortices and pathologic fracture was seen [figure 2a]. A multi - centric growth pattern was seen showing a permeative type of destruction [figure 2b]. (a) computed tomography (ct) showing a large multilocular osteolytic lesion extending from 37 to 47 region crossing the midline, destruction of both cortices and pathologic fracture are seen, (b) ct revealing a multi - centric growth pattern and showing a permeative type of destruction a chest radiograph was taken to rule out any primaries in the lung . A provisional diagnosis of ameloblastoma was established . An incisional biopsy was performed under local anesthesia and microscopic examination revealed odontogenic islands infiltrating the connective tissue, the peripheral tall columnar cells showed proliferation and peripheral palisading of basal cells with reverse polarity of the nucleus . (a) h and e, 10 low power view showing an odontogenic island with peripheral tall columnar cells showing proliferation and peripheral palisading of basal cells with reverse polarity of the nucleus, (b) h and e, 40 low high power view showing cells with atypical features of pleomorphism, hyperchromatism, altered nuclear - cytoplasmic ratio, and mitotic figures high power view showed cells with atypical features of pleomorphism, hyperchromatism, altered nuclear - cytoplasmic ratio, and mitotic figures [figure 3b]. A final diagnosis of ameloblastic carcinoma was established . Malignant epithelial odontogenic tumor, which includes the malignant ameloblastoma, ameloblastic carcinoma, primary intraosseous squamous cell carcinoma, clear cell odontogenic tumor, and malignant epithelial ghost cell tumor are very rare . Elzay, slootweg and mller used the term ameloblastic carcinoma to convey the presence of cytologic features of malignancy . The degree of differentiation in epithelial neoplasms is usually considered to be significant in predicting biologic behavior of metastasis . The main difference between elzay's and slootweg and mller's schemes relates to the minor point of histogenesis . According to these authors, the term ameloblastic carcinoma should be used to designate lesions that exhibit histologic features of both ameloblastoma and carcinoma . The tumor may metastasize and histologic features of malignancy may be found in the primary tumor, the metastases or both . The term malignant ameloblastoma should be confined to those ameloblastomas that metastasize despite an apparently typical benign histology in both the primary and the metastatic lesions . The incidence of ameloblastic carcinoma is greater than that of malignant ameloblastoma by a 2:1 ratio . The neoplasm may be derived from a number of different sources such as those of odontogenic origin, including ameloblastoma, odontogenic cysts or epithelial odontogenic rests as well as entrapped salivary gland epithelium or epithelium entrapped along embryonic fusion sites . The consensus now is to use the term ameloblastic carcinoma for those tumors with the histological evidence of malignancy in the primary, recurrent, or metastatic tumor regardless of whether there is metastasis or not although malignant ameloblastoma is reserved for metastasizing ameloblastomas, which exhibit benign histological features both in the primary and metastatic lesion . The characterization of carcinoma arising centrally within the mandible and the maxilla is an uncommon, but complex problem . The first step in the staging process must be the exclusion of metastasis or invasion of bone by tumor from adjacent soft - tissue or paranasal sinus . Carcinomas in the jaws metastasizing from primary locations such as the lung, the breast and the gastrointestinal tract may mimic ameloblastic carcinoma and must always be ruled out clinically before a diagnosis is made . The clinical and radiological picture most commonly resembles ameloblastoma, but ameloblastic carcinoma can be suspected if there is a sudden increase in the size of the swelling, pain, trismus, paresthesia and numbness or if there is any of foci of calcification as these features are unusual in ameloblastoma . In the present case, the swelling was large involving a considerable portion of the mandible extending from the left ramus to the right ramus . The mitotic features in ameloblastic carcinoma have been considered as significant by few authors especially when ameloblastic carcinoma is arising de novo, wherein mitosis is higher . In the present case, there was no evidence of regional or distant metastasis, but there was histological evidence of typical ameloblastic cells and anaplastic cells in the same tumor . In addition, there was cellular pleomorphism and nuclear hyperchromatism with occasional mitoses in the same tumor . Primary intra - alveolar epidermoid carcinoma must be considered in the differential diagnosis of ameloblastic carcinoma . Although, the primary intra - alveolar carcinoma and the ameloblastic carcinoma exhibit some clinical differences, their histologic features are similar enough to suggest a histogenetic relation . It is possible, then, that the primary intra - alveolar carcinoma may represent simply a less differentiated, usually non - keratinizing form of ameloblastic carcinoma, both lesions being derived from odontogenic remnants . Our case occurred in a 44-year - old female with involvement of both sides of the mandible . Thus, the term ameloblastic carcinoma can be applied to our case, which showed focal histologic evidence of malignant disease including cytologic atypia and mitoses with indisputable features of classic ameloblastoma . The treatment of and prognosis for ameloblastic carcinoma is unclear in the literature due to the rarity of this tumor and the lack of well - documented patients . Surgical excision, with or without adjuvant radiotherapy, seems to be required for local control . We have presented a rare case of a large ameloblastic carcinoma in a 44-year - old female, which involved a large considerable part of the mandible crossing the midline making this case unusual, which was treated by radical surgery and immediate jaw reconstruction . It has been suggested that the high rate of recurrence is due to its mode of growth and surgical mismanagement rather that any inherent malignant properties and metastases are exceedingly rare . The clinical and biological differences between conventional ameloblastoma and ameloblastic carcinoma are significant and can be useful to distinguish between the two entities when the pathological diagnosis is not certain.
A host of animal models have been proposed with relevance to modeling the pathological features which characterize alzheimer's disease (ad) brain . In order to test the validity of the animal models, properties and predictions from animal models these features involve a broad spectrum of altered properties from those of control nondemented (nd) individuals including the presence of enhanced deposits of amyloid- peptide (a), neurofibrillary tangles (nfts), abnormalities in vasculature [35], evidence for ongoing chronic inflammation [6, 7], and loss of neurons and synaptic connectivity . Advancement, fine tuning, or rejection of animal models involves a rigorous and complex comparative process concerning the testing of many variables . Intrahippocampal injection of a 142 in rat brain has been suggested as an animal model which emphasizes the inflammatory reactivity present in human ad brain . This model shows marked enhancement of microgliosis in response to peptide relative to control injections of pbs (phosphate buffered saline) vehicle or reverse peptide (a 42 - 1). In addition, hippocampal neuronal loss is significantly increased with a 142, compared to control, injection . Importantly, drug inhibition of microglial inflammatory responses has a demonstrated efficacy for conferring neuroprotection [1012]. Comparison at the molecular and cellular levels has included the finding that enhanced expression of the purinergic ionotropic p2x7 receptor in activated microglia occurred similarly both in the animal model and in ad brain tissue . Another feature of the a 142 rat model is evidence for an inflammatory response involving altered vasculature including a leaky blood - brain barrier (bbb). In this case a 142 induced an increased permeability of bbb compared to control pbs injection allowing infiltration of plasma protein into parenchymal brain regions . Elevated brain fibrinogen was suggested as an amplifying factor for microglial activation and inflammatory reactivity . Overall, the results from in vivo studies indicated that a 142 injection elicited microglial reactivity which may be associated with a weakened bbb . These findings enhanced creditability of the model for predicting vascular changes since evidence has suggested bbb in ad brain may be damaged . The overall purpose of the present study was to test and extend the utility of a peptide intrahippocampal injection as an animal model of ad . The present work was specifically designed to compare vascular perturbations in the animal model with specific microvessel changes and irregularities evident between ad and nd brain tissue . The comparison has included numbers and lengths of microvessels and abnormalities in microvessels including vessels with constricted diameters . All protocols involved studies on male sprague - dawley rats and were approved by the university of british columbia animal care ethics committee . The relevance of using intrahippocampal a 142 as an animal model of ad has been reviewed, and the overall procedures employed in use of the model have been detailed in previous work from this laboratory [1013]. Briefly, stereotaxic injection of peptide (2 nmol of full length a 142; california peptide, napa, ca) or control (pbs) was performed into the dentate gyrus region of hippocampus (ap: 3.3 mm, ml: 1.6 mm, dv: 3.2 mm). Injection of peptide, compared with pbs, precipitates an enhanced microglial inflammatory response accompanied by substantial loss of hippocampal neurons . At one week postinjection brains were removed and postfixed and cryoprotected prior to cutting into 40 m sections . Free - floating sections were processed for immunohistochemistry with sections incubated overnight at 4c with primary antibodies to rat endothelial cell antigen (reca-1, 1: 1000; serotec, oxford, uk) or laminin (1: 1000; sigma, st . Sections were incubated in alexa fluor conjugated secondary antibodies (1: 200; invitrogen, carlsbad, ca) for immunofluorescence staining . In immunostaining controls, primary antibody was omitted in all staining procedures . The tissues were examined under a zeiss axioplan-2 fluorescent microscope (zeiss, jena, germany) using a dvc camera (diagnostic instruments, sterling heights, mi) with northern eclipse software (empix imaging, mississauga, on, canada). The digitized images were analyzed using nih imagej 1.37 b software (national institute of health, bethesda, md). Four coronal hippocampal sections (200 m apart) were used for quantitative analysis (detailed procedures in [1013]). In each stained section, four nonoverlapping fields within the granule cell layer and molecular layer regions were selected (magnification of 40x). Density and lengths of microvessels were measured in molecular layer regions of dentate gyrus; mean values for these variables were found from averaged values over 1.3 mm areas . In some experiments numbers of microvessels with narrow diameters were measured . Postmortem medial temporal cortical (mtc) tissue was obtained from the kinsmen laboratory brain bank at the university of british columbia (vancouver, bc, canada). Protocols for use of human tissue were in accordance with ethical guidelines established by the university of british columbia . Brain tissue from mtc was obtained from 9 nd cases (7599 years of age; mean 83.0 2.5) and 9 ad cases (6590 years of age; mean 76.2 2.8). Neuropathological criteria were used in the classification of brain tissue with nd cases showing no clinical or pathological history of dementia or other neurological disorders . Ad cases were characterized by immunohistochemical assessment of the density of plaques and nft in limbic and neocortical areas (including hippocampus, amygdala, and frontal, temporal, parietal, and occipital cortex). All ad cases conformed to criteria provided by the national institute on aging and reagan institute . The protocols used for laminin immunohistochemical staining in ad and nd brain tissue have been detailed [12, 16]. Briefly, free - floating sections with 40 m thickness were cut from medial temporal cortical tissue . Sections were then transferred into pbs / triton - x solution and incubated overnight at room temperature with laminin (1: 1000, sigma). Biotinylated secondary antibody was applied followed by incubation in avidin - biotinylated horseradish peroxidase complex with labeling visualized by incubation in diaminobenzidine (dab) solutions . Sections were then washed and mounted on glass slides, air - dried, and coverslipped; immunostaining controls were performed using standard procedures omitting primary antibodies; under these conditions no control staining was observed for any marker . The images were acquired using a light microscope (olympus bx51) and a digital dp71 camera (olympus, center valley, pa). Four sections in the grey matter of medial temporal cortex were used for analysis with fields separated by fixed distances to ensure no overlap between image areas . Laminin staining was used to measure numbers and lengths of microvessels in 1 mm cortical areas . Constricted microvessels, with diameters 3 m, were also measured in the cortical brain regions . The digitized images were analyzed using nih imagej 1.37 b software (national institute of health, bethesda, md). Statistical significance was assessed by one - way anova, followed by student - newman - keuls multiple comparison test (graphpad prism 3.0). Typical patterns of microvessel staining (reca-1 marker) are shown for control pbs vehicle (figure 1(a), left panel) and a 142 (figure 1(a), right panel) at 7 d after injection in rat hippocampus . The area of staining was for the molecular layer of dentate gyrus . Although morphological differences were observed between the two animal groups (see below), the most evident difference was the increased number of microvessels in peptide - injected brain . The numbers of microvessels / mm for control and a 142 are presented in the bar graph of figure 1(b). Also shown are the mean lengths of microvessels for the two animal groups (figure 1(c)). Overall (n = 5 each for control and a 142 group), peptide - injected brain demonstrated a 42 7% increase in numbers of microvessels, normalized to area, compared to control pbs injection . Mean length of microvessels was significantly decreased (by 27 4%) in a 142, compared with pbs, injection . Although the increase in microvessel density could indicate angiogenesis, it should be noted that a specific angiogenic marker would be required to assess formation of new blood capillaries in peptide - injected brain . Inspection of the morphology of microcapillaries indicated enhanced irregularities in vessels from peptide - injected hippocampus compared with control (figure 2). For example, fragmented microvessels were much more common after a 142 compared with pbs, injection . Other microvessel abnormalities which were relatively abundant following peptide injection included constricted microcapillaries (see below) and microvessels with looping, knob - like, and uneven appearances (figure 2). Although these morphological categories largely reflect subjective descriptions, such characteristics were not commonly observed in pbs - injected rat hippocampus . These narrow microcapillaries (laminin staining, figure 3(a)) exhibited 3 m or less in diameters and often showed attachment to larger width microvessels . In some cases the constricted microcapillaries formed a bridge between adjacent larger blood vessels . Overall (n = 5, figure 3(b)), 14 3.1% of microvessels in a 142-injected rat hippocampus demonstrated constricted diameters; the corresponding value in pbs - injected brain was 4 1.4% . At present, it is not known if the constricted microvessels represent angiogenic vessels or possibly a population of damaged vessels . Representative laminin immunoreactivity (ir) for microvessels from the two categories of cases is presented in figure 4(a) (nd, left panel; ad, right panel). Microvessels in nd showed a typical pattern of linear shape with little evidence for multiple branching from the primary vessel . A different pattern of microvasculature was manifest in ad tissue with microvessels commonly exhibiting considerable variability in length and diameter . In particular, ad tissue commonly demonstrated microvessels with short lengths (fragments) and sections with narrow and constricted diameters (see below); such properties were largely absent in nd cases . The results (figure 4(b)) show that microvessel number was 72 9% higher in ad, relative to nd, brain (n = 9 cases each for nd and ad); this difference was significant . Mean length of microvessels was significantly lower, by 18 5%, in ad, relative to nd, tissue (figure 4(c)). A pattern of altered microvessel morphology was observed between vasculature in ad and nd brain tissue . In particular, ad microvessels demonstrated a considerable variation of morphological shapes including thin fragments and nonlinear segments . Fragmented and narrow (see below) microvessels were particularly evident in ad tissue with vessels exhibiting ring - like morphology . Additionally, ad microvessels often presented as uneven diameters with areas of dilation and formation of knob - like structures . Although nd cases demonstrated microvessels with similar features to those described in ad tissue, their frequency of appearance was scarce . Previous work has documented morphological features and abnormalities in microvessels in tissue obtained from ad individuals [1719]. A common finding in ad cases was the appearance of constricted microvessels in the temporal cortex as shown in the representative laminin staining (arrows, figure 6(a)); examples of constricted microvessels were much less evident in nd cases . As for the animal model, microvessel constriction was defined as a diameter equal to, or less than, 3 m . Overall (nd, n = 9; ad, n = 9), constricted microvessels in nd cases comprised only 3.9 2.2% of total vessels (figure 6(b)). Thus microvessel constriction appears as a prominent characteristic in ad brain, a distinguishing feature differentiating vasculature in diseased tissue from nd cases . The primary aim of this work was to compare microvessel number, length, and morphological properties between a 142 and control pbs intrahippocampal injection in a rat model of ad with the same variables in human ad and nd brain tissue . The overall results show a similar pattern of microvessel perturbations in a peptide - injected rat brain as present in ad brain . The changes include an increased mean number and diminished mean length of microvessels and prominent expression of microvessel abnormalities in peptide - injected rat and in ad brain tissue compared with pbs - injected and nd brain . The abnormal properties include the presence of constricted microvessels which were largely absent in controls . These results extend previous findings that the a 142 animal model exhibits characteristics similar to those in ad brain including an altered bbb permeability, enhanced inflammatory reactivity, and neuronal loss . The increased density of microvessels in a 142 versus pbs (rat) and ad versus nd (human) could suggest angiogenic activity in ad brain . However, a proviso is that a specific marker for angiogenesis is not well defined . Although increased expressions of laminin [20, 21] and integrin v3 [22, 23] have been suggested as indicators of angiogenesis they are not specific markers which differentiate between newly formed and existing capillaries . Nevertheless, a consistent enhancement in microvessel number was a common pattern of change with a 142 injection (versus pbs in the animal model) and in ad (versus nd) brain tissue . It is possible that angiogenic activity could be associated with increased leakiness of blood vessels . In this regard, extravasation of plasma proteins including albumin has been measured in the peptide - injected rat hippocampus . The possibility of angiogenesis in ad brain is supported by the finding that a spectrum of proangiogenic factors is elevated in ad pathology . Increased levels of vegf are reported in microglia obtained from ad patients and in human microglia exposed to a 142 . In addition, app23 transgenic mice show increased formation of new vessels which was inhibited using an antagonist for vegf . Taken together, particular morphological irregularities were evident in a 142-injected dentate gyrus and ad brain tissue and largely absent with pbs injection and in nd brain . These abnormalities were characterized as fragmented, looping, knob - like, uneven, and constricted . Typical representations of these features are shown for a 142 animal treatment (figure 2) and for human ad cases (figure 5). Since constricted microvessels (diameters 3 m) were clearly definable compared with the other abnormal properties, it was possible to quantify their expression . Overall, about 4% of total vessels in controls were constricted compared with values of 14% in a 142-injected rat hippocampus and 23.6% in ad brain . It can be noted that other morphological abnormalities, such as small microvessel fragments, were evident in diseased tissue . The presence of fragments could underlie the diminished mean length of microvessels in ad brain . The altered morphology of microvessels suggests concomitant changes in cerebral blood flow in intact animals . Future studies are required to determine the effects of a 142 intrahippocampal injection on the hemodynamics of cerebral blood flow . Recent work using transgenic animals expressing elevated a 142 has reported impaired cerebral autoregulation of flow which is intact in human ad subjects [27, 28]. These studies also point out that extrapolation of animal results to describe similar processes in humans requires considerable caution . Overall, this work provides evidence that an animal model of a 142 intrahippocampal injection reproduces some microvessel perturbations which are present in ad brain . Combined with measurements on bbb dysfunction, the a 142 animal model has demonstrated efficacy in simulating prominent vasculature changes evident in diseased tissue . Future testing of other abnormal processes characteristic of ad brain such as synaptic dysfunction will help assess the overall validity of the ad animal model . In addition, prolonged exposure of brain microenvironments to a 142 for durations in excess of 7 d will be relevant to testing the animal model for altered vasculature and microglial - vascular interactions under conditions of chronic inflammation.
The sodium - glucose cotransporter-2 (sglt-2) inhibitors are a novel class of glucose - lowering drugs which act by inhibiting the reabsorption of filtered glucose from the kidneys . This effect is mediated by inhibition of sglt-2 co - transporters, which are expressed in the proximal renal tubule . While their predominant mode of action is renal, sglt-2 inhibitors appear to have effects on other parts of the glucose homeostatic system as well . Clinical data suggest an improvement in beta - cell function, perhaps mediated by reversal of glucotoxicity, and a reduction of insulin resistance, with sglt-2 inhibition . Recent data, from studies performed with sglt-2 inhibitors, have demonstrated a fall in insulin to glucagon ratio upon acute exposure to these drugs; the change is the ratio is contributed to increase in the glucagon and fall in the insulin level concentration . This change persists, though to an attenuated degree, even after 28 days of sglt-2 inhibitor administration . In 1966, the bihormonal hypothesis, proposed by unger, clearly highlighted the important role of glucagon in the pathophysiology of diabetes . Developments in this field, however, were overshadowed by insulin - centric research, though a few experts did highlight its significance . The inclusion of the alpha cell in de fronzo's infamous ominous octet, coupled with advancement in therapeutics such as glucagon receptor antagonists, and bihormonal bionic (two hormone) artificial pancreas devices, has brought glucagon back to the center stage of diabetes research . While type 1 diabetes subjects manifest an exaggerated glucagon response to arginine, mediated by severe intra - islet deficiency of insulin, persons with type 2 diabetes may have alpha - cell resistance to insulin, which prevents insulin (whether endogenous or exogenous) from suppressing glucagon release in response to arginine . The insulin glucagon ratio (igr) was a relatively frequently cited index in the literature from the 1970s and 1980s . Igr was assessed in various conditions, including nonglycemic states like burns and starvation, and dysglycemic syndromes including type 2 diabetes and gestational diabetes mellitus . Insulin glucagon ratio can be used as an index of anabolism, insulin is the most potent anabolic hormone in the body, and a high igr reflects its action, as opposed to that of glucagon, which has glycogenolytic or catabolic activity in the liver ., this may be because of the development of more accurate indices and parameters which measure insulin sensitivity / resistance . The challenges posed by recent understanding of sglt-2 inhibition, however, have created interest in this index . There is, however, an associated rise in endogenous glucose production, noted both after acute and chronic administration of sglt-2 inhibitor, which attenuates the glucose - lowering efficacy of the drug . Group suggest that this paradoxical response is a manifestation of an adaptive physiological response to loss of calories in the urine . We discuss the utility of the igr in current diabetes management, and suggest three hypotheses in addition to those put forward by bonner et al . And ferrannini et al ., to explain the effect of sglt-2 inhibition on the index . Though no firm conclusions can be drawn from current data, available knowledge opens up exciting vistas for further research . Recently, the presence of sglt-2 receptors in the alpha cell has been reported by bonner et al . The authors suggest that sglt-2 expression in alpha cells, when down - regulated, is associated with an increase in the expression of sglt 1 and glucagon genes, as well as genes related to hepatic gluconeogenesis . The same research team reports that sglt 1 and 2 gene expression is lower in islets of type 2 diabetes as compared to normal subjects . Similar results are obtained when slcsa-2 gene is knocked out, or when it is inhibited with dapagliflozin . This may explain the paradoxical increase in plasma glucagon and hepatic glucose production noted with sglt-2 inhibition . The workers who have reported a fall in igr upon exposure to sglt-2 inhibitor, propose correction of hyperglycemia, and alleviation of glucotoxicity upon the alpha cell (mediated perhaps by paracrine effects), as a mechanism for the relative hyper - glucagonemia . Sodium - glucose cotransporter 2 inhibition leads to lowering of plasma glucose, which in turn brings counter - regulatory processes into motion . The first line of defense against hypoglycemia is a reduction in insulin levels, while the second line is an increase in glucagon, mediated by intra - islet sensing of glucose . The third line of defense, an increase in epinephrine release, is mediated by peripheral and central nervous system sensing . Though plasma glucose concentrations with sglt-2 inhibition do not fall to hypoglycemic levels, or to levels which are thought to stimulate glucagon secretion (70 mg / dl), it is possible that relatively low intra - islet glucose concentrations or low intra - islet insulin release, have a paracrine effect upon the alpha cells . Sglt-2 inhibitors may increase the alpha cell's glucose sensitivity through receptor - dependent, or through receptor - independent modes of action . The latter may be a mechanism of action distinct from the alpha cell sglt-2 receptor - based pathway suggested by bonner et al . The lowered igr suggests a catabolic state, or more accurately, mimicry of calorie restriction . This may be construed as a beneficial action, designed to correct the maladaptive anabolism noted in obese type 2 diabetes . The lowering of igr with sglt-2 inhibitors may explain or may be a corollary of, weight loss noted with these drugs . After 4 weeks, chronic treatment with sglt-2 inhibitors total glucose disposal was reduced due to prolonged reductions in insulin and glucose with maintained energy expenditure . The enzyme carnitine palmitoyl transferase 1 (cpt 1) acts as a gateway to the mitochondria to help allocate free fatty acids (ffas) to either of two pathways: -oxidation, or conversion to triacylglycerol . It is possible that sglt-2 inhibition may stimulate cpt 1, perhaps by suppressing malonyl coenzyme a (coa) (a potent inhibitor of cpt 1), and diverts ffas to -oxidation, instead of fat deposition . This enzyme has been identified at the site of action of other classes of oral anti - diabetic drugs and will be discussed further . In preclinical animal models, as sglt-2 inhibitor treatment is known to increase plasma ffa levels, further research is warranted on effect of ffa on alpha cell with the use of sglt-2 inhibitors . Recent data which describe a reduction in igr after sglt-2 inhibitor empagliflozin treatment administration uses estimated prehepatic igr calculated from plasma concentrations of insulin and glucagon . It is possible that concentrations of the hormones and their gradient may differ if estimated through portal vein sampling . Recently, the presence of sglt-2 receptors in the alpha cell has been reported by bonner et al . The authors suggest that sglt-2 expression in alpha cells, when down - regulated, is associated with an increase in the expression of sglt 1 and glucagon genes, as well as genes related to hepatic gluconeogenesis . The same research team reports that sglt 1 and 2 gene expression is lower in islets of type 2 diabetes as compared to normal subjects . Similar results are obtained when slcsa-2 gene is knocked out, or when it is inhibited with dapagliflozin . This may explain the paradoxical increase in plasma glucagon and hepatic glucose production noted with sglt-2 inhibition . The workers who have reported a fall in igr upon exposure to sglt-2 inhibitor, propose correction of hyperglycemia, and alleviation of glucotoxicity upon the alpha cell (mediated perhaps by paracrine effects), as a mechanism for the relative hyper - glucagonemia . Sodium - glucose cotransporter 2 inhibition leads to lowering of plasma glucose, which in turn brings counter - regulatory processes into motion . The first line of defense against hypoglycemia is a reduction in insulin levels, while the second line is an increase in glucagon, mediated by intra - islet sensing of glucose . The third line of defense, an increase in epinephrine release, is mediated by peripheral and central nervous system sensing . Though plasma glucose concentrations with sglt-2 inhibition do not fall to hypoglycemic levels, or to levels which are thought to stimulate glucagon secretion (70 mg / dl), it is possible that relatively low intra - islet glucose concentrations or low intra - islet insulin release, have a paracrine effect upon the alpha cells . Sglt-2 inhibitors may increase the alpha cell's glucose sensitivity through receptor - dependent, or through receptor - independent modes of action . The latter may be a mechanism of action distinct from the alpha cell sglt-2 receptor - based pathway suggested by bonner et al . The lowered igr suggests a catabolic state, or more accurately, mimicry of calorie restriction . This may be construed as a beneficial action, designed to correct the maladaptive anabolism noted in obese type 2 diabetes . The lowering of igr with sglt-2 inhibitors may explain or may be a corollary of, weight loss noted with these drugs . After 4 weeks, chronic treatment with sglt-2 inhibitors total glucose disposal was reduced due to prolonged reductions in insulin and glucose with maintained energy expenditure . The enzyme carnitine palmitoyl transferase 1 (cpt 1) acts as a gateway to the mitochondria to help allocate free fatty acids (ffas) to either of two pathways: -oxidation, or conversion to triacylglycerol . It is possible that sglt-2 inhibition may stimulate cpt 1, perhaps by suppressing malonyl coenzyme a (coa) (a potent inhibitor of cpt 1), and diverts ffas to -oxidation, instead of fat deposition . This enzyme has been identified at the site of action of other classes of oral anti - diabetic drugs and will be discussed further . In preclinical animal models, as sglt-2 inhibitor treatment is known to increase plasma ffa levels, further research is warranted on effect of ffa on alpha cell with the use of sglt-2 inhibitors . Recent data which describe a reduction in igr after sglt-2 inhibitor empagliflozin treatment administration uses estimated prehepatic igr calculated from plasma concentrations of insulin and glucagon . It is possible that concentrations of the hormones and their gradient may differ if estimated through portal vein sampling . Sodium - glucose cotransporter-2 inhibitors can be compared and contrasted with existing classes of glucose - lowering drug . Glibenclamide has been shown to inhibit the cpt 1 gateway, thus diverting substrate toward the triacylglycerol production . Metformin, on the other hand, activates the cpt 1 gateway . By inactivating acetyl coa carboxylase, which catalyzes the biosynthesis of malonyl coa this molecule acts as a substrate for de novo fatty acid biosynthesis and inhibits cpt 1 . Lower malonyl coa production allows the cpt 1 pathway to divert ffa to the -oxidative pathway . It is possible that sglt-2 inhibitors act in a manner similar to that of metformin in addition to weight loss due to calories lost in the urine . It must be mentioned here that metformin is a calorie restriction mimetic, with geroprotective effects . Whether, currently available sglt-2 inhibitors share similar properties remains the focus of future research . At the same time, it is prudent to clarify that the cpt 1 enzyme is not a homogenous entity . It has multiple isoforms, expressed in different organs, and much needs to be learnt about its biology . Another similarity of sglt-2 inhibitors with metformin is that they are able to achieve incretin enhancement without causing hyperinsulinemia . This property contrasts with that of incretin - based therapies such as dipeptidyl peptidase 4 inhibitors and glucagon - like peptide-1 analogs . It supports a multifaceted effect of sglt-2 inhibitors, beyond inhibition of co - transporter in proximal renal tubule and proposes that these drugs will be able to shift the body milieu from maladaptive anabolism in type 2 diabetes to adaptive metabolism . The igr should also be studied as a tool for research and clinical decision - making . Inclusion of plasma glucagon values or portal glucagon concentrations, in formulae or models created to measure insulin sensitivity, may improve their utility.
The forensic age estimation of unidentified skeletons and corpses for the purpose of identification has been a conventional feature of forensic science . Determining the identity of a decedent is of considerable significance from the ethical, legal, and criminal perspectives; not only is it the prerequisite for officially declaring an individual dead but it is also the basis for dealing with mass disasters, crimes, and war crimes.1 compared to bone mineralization, tooth mineralization stages are much less affected by variation in endocrine and nutritional status, and developing teeth therefore provide a more certain indication of chronological age.2 tooth formation is used often to assess maturity and predict age . Within clinical dentistry, this information aids in diagnosis and treatment planning . In forensic odontology and archaeology, age estimation methods can aid the identification of age at death of a deceased child and also give important information with regard to past populations . Age estimation is also proving valuable when birth data is lacking or doubted in the management of immigration to help determine physiological age.3 the scientific basis of age estimation is the genetic control of ontogenesis, which delimits the temporal variation of developmental stages.4 according to the suggestions produced by the study group on forensic age diagnostics,1 a forensic age estimate of a living person for the purpose of criminal prosecution should consist of: a physical examination that also records anthropometric data, any age - relevant developmental disorders and signs of sexual maturation; an x - ray examination of the left hand; and a dental examination that records dentition status and evaluates an orthopantomograph . Several methods for the determination of dental development from radiographs have been described.5 in children and adolescents, age estimations are based on the developmental stage of the deciduous and permanent dentition.6 most of these are based on a comparison of the radiographic development of teeth with standard diagrams collected from a large number of persons, usually in a well - defined geographic region.7 with advancing age, secondary dentine is deposited along the wall of the dental pulp chamber, leading to a reduction in the size of the pulp cavity.8 tooth wear has been widely used as a tool of age estimation.9 the mechanisms by which teeth wear include attrition, abrasion, and erosion . Tooth attrition is defined as gradual and regular loss of tooth substance as a result of chewing activity.10 age - related changes can be evaluated from dental radiographs.11 this study aimed to determine possibility of age estimation with the orthopantomographs (opgs); the difference between the evaluations of opgs by dentists and the decade provide the most accurate age estimation . In this retrospective study, panoramic radiographs of 238 turkish people of known chronological age, with ages ranging from 1 to 60 years, were enrolled . The opgs taken at the 19 mays university, faculty of dentistry, department of oral diagnosis these opgs were evaluated by two independent dentists at the same time interval . The age estimation for each case was done according to decade . Then the estimation results were compared to the chronological age of individuals by third independent dentist . Two correct estimations were recorded as true - true, false estimations recorded as false - false, and one correct and one false result were recorded as correct - false . The opgs were selected according to the criteria12 listed below: both mandibular branches had to be of equal widththe curve had to appear concavethe depth of field should be given both mandibular branches had to be of equal width the spee curve had to appear concave the depth of field should be given patients were classified: group 1: 110 yearsgroup 2: 1120 yearsgroup 3: 2130 yearsgroup 4: 3140 yearsgroup 5: 4150 yearsgroup 6: 5160 years the evaluation criteria for the opgs for both of dentists were compiled from the literature.13,14 presence of primary teeth in mouth mixed dentition period presence of third molar teeth in mouth apexogenesis and maturation stage of third molar teeth enamel attrition level of teeth width of root canal and pulp cavity level of alveolar bone resorbtions were considered during age estimation the number and ratio of true and false predictions of two dentists were evaluated, and similarities and/or differences among them were calculated using a chi - square test . Among the 238 opgs evaluated for age estimation, the first dentist predicted 174 of the cases (73.1%) orthopantomographs correctly and 64 of the cases (26.9%) incorrectly . The second dentist predicted 171 of the cases (71.8%) correctly and 67 of the cases (28.2%) incorrectly . The two clinicians evaluated 238 radiographs and predicted 149 of the cases (62.9%) correctly and 42 of the cases (17.6%) incorrectly . While evaluating the opgs together, 47 of the cases (19.8%) were evaluated correctly by one dentist and incorrectly by the other . The percentage of true and false age estimations of both dentists is shown in figure 1 and table 1 . There was no significant difference between the dentists in terms of age estimation (p>.05), and the accuracy of age estimation in the age groups was found to be high (=0.73). The greatest accuracy in the age estimations of the dentists occurred in the range of 110 years of age (89.6%), and the greatest percentage of incorrect age estimations occurred in the range of 4150 years of age (41.7%). The correct age estimation percentage diminished in age estimation plays an important role in forensic medicine, clinical dentistry, pediatric endocrinology, and archaelogy.15 age estimation is of wider importance in forensic medicine, not only for the purpose of identifying deceased victims but also in connection with crimes and accidents . In addition, chronological age is important in most societies for school attendance, employment, social benefits, and marriage.2 in adulthood, teeth undergo time - related changes representing biological aging, and many studies have shown that several features of aging can be used for age determination.12 gustafson6 developed the first dental method of age estimation in adults based on six criteria: attrition, secondary dentine in the pulp, cementum annulations, root resorption, periodontal recession, and root translucency . Furthermore, opgs also provide information regarding individuals identity16 and other age - related features such as enamel attrition, secondary dentine in the pulp, root resorption, cementum annulations, and periodontal recession.13 in our study, the age groups were determined and age estimation was made in accordance with these groups . In the present study, considering the necessity of identification and accurate age estimation of cases in daily practice of forensic medicine (age estimation for marriage, penal trials, and legal actions), and in the identification of disaster victims, a subjective age estimation method was implemented by evaluating opgs . Tooth improvement is a continuous process, but determining the end point of tooth development is very difficult . Thus, the calculation of a mean age for each phase is difficult; further research is needed to determine the apex closure stage of teeth.17 measurement using dental radiographs may be useful as a non - invasive technique for estimating the age of adults, both living and dead, in archaeological studies and in forensic work, but the method should be tested on an independent sample.18 a scoring system for age estimation should be developed to ensure good reliability . To minimize the influence of intra - and inter examiner variations, well - defined criteria and careful calibration among examiners are esential.19 even though the coronal pulp might be affected more by external factors than the root pulp, the scoring systems gave opgs a strong correlation between age and the amount of secondary dentin for the coronal and for measurements in the root area . Age estimations based on measurements of the amount of secondary dentin present seem to be relatively reliable.20 methods of age estimation in adults are concerned, and in view of the relative accuracy of the age estimations performed, one should keep in mind that the standard deviations of such age estimations are, in general, about 10 to 12 years.2 in this study, a scoring system was not used . Age estimation was made using criteria such as the presence of primary teeth in the mouth, mixed dentition period, presence of third molar teeth in the mouth, apexogenesis and maturation stage of third molar teeth, enamel attrition level of teeth, width of root canal and pulp cavity, and level of alveolar bone resorbtions . Bosmans et al,14 using opgs in a study, selected 6 teeth and performed age estimation using criteria such as maximum tooth length, pulp length, and root and pulp width . They classified the study population, whose ages ranged from 29 to 70 years, into decades with time intervals of 9 years . The age estimations were reported to be convenient to the decades of chronological ages, based on a single dentist s evaluation . The criteria used in our study were a compilation derived from the literature; however, the manner of evaluation in this study was different . The authors were inspired by previous studies related to the subject in order to take a further step . Opgs were evaluated considering the age groups of the study population, which is concordant with the previous studies performing evaluation according to decades.12 in parallel to the literature, author should suggest the use of skeletal measurements in addition to dental methods, for accurate age estimation.7 despite the variations related to the practitioners, in this study, there were no significant differences in age estimation between both two participant practitioners . Age estimation through the evaluation of opgs revealed the most reliable results for the first decade of life . The possible reasons might be the reduction of the criteria and signs for the age estimation of opgs in older persons and the variability of the oral health status of patients in older age groups . Age estimation with opgs can be used to make a significant percentage of forecasts in areas such as forensic medicine and forensic dentistry, especially in young patients . In order to achieve accurate and reliable age estimation, in addition to millimetric measurements of the teeth, skeletal measurements and examinations
The relationship between mind and matter has been a fundamental topic of investigation in many if not all cultures and traditions since the most ancient records of human thought, from the hindu orthodox school of sankhya nearly 27 centuries ago to the classic greek philosophy of plato (e.g., in the dialogue phaedo) 300 years later . With few exceptions (most noticeably that of panpsychism:) most theories of the mind throughout history related it to the body and its various parts, including the heart in aristotle's view and the endocrine pineal gland in the work of descartes . Early physicians hippocrates and galen were among the first influential proponents of the central role of the brain in the operation of the mind based on anatomical and physiological observations . The development of modern neuroscience led to the (still ongoing) accumulation of massive evidence that irreversibly linked the mind to the brain . The goal of this spotlight paper is emphatically not to provide an extensive review or even a balanced perspective of the enormous body of work on the brain - mind relationship in cognitive philosophy . To appreciate the breadth and depth of this topic, we refer the reader to a sample collection of over 200 articles on consciousness and neuroscience available online . Instead, we selectively review a set of specific topics in neuroscience and cognitive science together supporting the notion that, within a particular interpretation, certain aspects of the mind - brain relationship can be framed as a rigorously defined and in principle soluble mathematical problem . In order to build this argument, we first describe in the next two sections the somewhat delimited facets of the mind and the brain we aim to bridge together . Next, we explicate what in our view would count as solving the mind - brain problem . Then we elaborate on relevant general topics in the investigation of the brain and of the mind that will likely play a central role in a satisfactory explanation of the mind - brain relationship . Finally, we overview a sample of more specific available frameworks in neuroscience and cognitive science that appear particularly promising among the known existing candidates to help crack the mystery of the link between mental and neural activities . The term mind is commonly employed to signify a broad variety of connotations even within the scientific discourse [10, 11]. When referring to the mind - brain relationship, mind is most often taken to stand for human consciousness . Consciousness is itself challenging to define, which may be viewed as a puzzling paradox, considering that it constitutes perhaps the most immediately and intimately accessible characteristic of the life of every person . Consciousness cannot even be taken as a trivial or necessary feature that is always present in our existence, because we all experience the transition of fading into dreamless sleep and awaking from it tens of thousands of times in the course of a typical life span . The distinction between inner mental content and outer behavioral observables is equally apparent when considering the obvious difference between dreamless sleep and a vivid dream . Judging from the paralyzed body (except for eye movement), an external spectator could not even begin to guess the mental state of a dreaming person . In contrast, we often toss, turn, kick, twitch, mumble, and moan during dreamless sleep . Here and in the rest of this paper we do not consider the legal or medical definitions of being conscious as opposed to unconscious, because the shared goal of physicians and lawmakers is to evaluate other people rather than themselves . Thus, they must necessarily take an objective, third - person perspective, that is, one entirely based on behavioral evidence . In contrast, what makes mental states so hard to relate to the brain, the body, and matter in general is precisely their being felt from within . Similarly, there are clear resemblances between the properties of consciousness and those of attention . Neuroscience has advanced considerably in the elucidation of the neural mechanisms underlying attentive processes (e.g.,). Nevertheless, recent evidence clearly indicates a double dissociation between attention and consciousness . Excluding the immediately cyclical definitions (such as equating consciousness to being aware and awareness to being conscious), one of the simplest approaches to describe consciousness is to define it as what it feels like being . Thus, a particular conscious sensation (such as the taste of chocolate) or thought (such as remembering someone's name) would just correspond to what it feels like to taste chocolate or what it feels like to remember that person's name . The challenge of the mind - matter relation, then, is to understand how particular configurations of matter (e.g., a person's body or brain) or material processes (the dynamic activity of that body or brain) systematically coexist or cooccur with feeling particular mental states . The ultimate hard problem is to explain why any material entity should feel like anything at all . Many aspects of conscious experience can be reported explicitly by the subject [22, 23] either internally, by inner dialogue or memory rehearsal, or externally, such as by a verbal description or numerical rating . A defining characteristic of such declarative content of the conscious mind is that it can be communicated . The most common form of communication among human beings is through spoken or written language, but declarative content can also be communicated by other means, such as body gesture, drawings, movies, and many others . Signifiers used (e.g., words and sentences in language), the declarative content they communicate is what we call their meaning . The field of semantics studies meaning and its communication by signifiers . Consider a subject experiencing a certain declarative mental state (e.g., anger) and communicating it to another person a fascinating facet of declarative communication is that the content intended by the sender is generally not identical to that understood by the recipient (e.g., why and how seriously is s / he angry?). Overwhelming scientific data unequivocally link the conscious mind to the brain, as opposed to matter at large . The general consensus on this interpretation in the research community boosted the popularity of the materialist credo that the mind is what the brain does . Available evidence, however, is more specific than generically pointing to the brain as the most relevant organ of the body in relation to the mind . Particular regions of the brain are more directly and intimately involved with mental activity, including the thalamocortical system and the hippocampal complex . Other parts of the central nervous system, such as the cerebellum and the spinal cord, appear less pertinent to the declarative content of the mind . The majority opinion is that what renders the parts of the brain relating to the mind special is not the intrinsic substance they are made of but their functional organization . In fact, those same thalamocortical and hippocampal regions are definitely active even during dreamless sleep, but their activity is different from that during conscious states . Thus, it is the information process, or computation, carried out by these brain structures, that most closely corresponds to consciousness [28, 29]. Implementing that same computation in different mediums, in artificial machines, or in virtual environments, would then in principle also result in conscious experience . Unfortunately, however, what might in fact constitute the essence of this computation, or its neural implementation, is far less clear . Proposals range from involvement of broadly distributed oscillatory rhythms (; see, however,) to activation of highly specific, we do not need to dwell into specific hypotheses, except noting a general consensus supporting spatial - temporal patterns of neural activity as key material correlates of conscious experience . The carriers of neural activity are neurons, biological objects each occupying a physical volume in the brain . Thus, the complete electric and chemical record of every small portion of each neuron and their connections in the entirety of the appropriate brain networks throughout the course of an arbitrarily large number of declarative mental states would in principle include all the necessary information about the brain that is relevant to the content of the mind . Within the particular framework we have chosen to adopt, the key players of the mind - brain relationship can be summarized as the following . On the one hand, each of us has access to the immediate experience of what it feels like to be, in any one moment of our conscious existence and throughout our life span . We can communicate that experience to each other, and our mental states are altered by receiving those messages . On the other hand, the brain is a dynamical information processing system, consisting of a large number of neurons, each characterized at any one time by the spatial distribution of its electrochemical state . Within a brain, neurons continuously exchange signals with each other, mutually affecting their spatially distributed electrochemical state . In this framework, why and how do certain spatio - temporal patterns of neural dynamics relate to declarative mental states? In other words, why do those neural dynamics that feel like something feel like those (or any!) Experiences? Many relationships are routinely observed in the course of everyday life, such as how the weight of bodies relates to their size or how satisfying it feels to drink when thirsty . Why is the mind - brain relationship so different as to constitute a problem? The main challenge seems to be that typically related observables are somehow of the same kind . For example, size and weight are both physical attributes of material objects, while thirst and satisfaction are both subjective sensations of minds . Brains and minds are so different from each other as to inspire several kinds of dualist philosophies in the course of time . This fundamental difference has been considered challenging by scientists and philosophers alike, from confronting possible inconsistencies in temporal dynamics through a retreat to the acceptance of limited explanations, to overt calls for giving up scientific accounts altogether . The more extreme positions maintaining that there is no solution to the mind - brain problem have been refuted both on philosophical and scientific grounds . The key issue is in fact to understand what would count as a solution . In other words, if the mind - brain relationship can be explained, what type of explanation is being sought? To answer this central question, it is useful to consider previous scientific breakthroughs that resulted in satisfactory understanding of relationships that had been considered difficult to explain before . The most illustrative examples would concern discoveries relating (sets of) observations that appeared completely disparate from each other, linking phenomena seeming of different kinds . A prime case in point is the explanation of thermodynamics in terms of statistical mechanics . The 1687 publication of newton's principia consolidated a century of gains from the scientific method introduced by galileo into a coherent and complete theory of mechanics . Newton's famous laws relating force to acceleration and defining the mutual attraction of masses by gravity provided an excellent description of planetary motion as well as accurate predictions of interactions among material objects . More than one hundred years later the first comprehensive treaty on thermodynamics was published, describing the relationship between heat, energy, temperature, and what was later called entropy . Thermodynamics appeared completely independent of newton's mechanics which explained instead how bodies move and respond to forces . While both mechanics and thermodynamics were (and still are) recognized as landmark advancements of scientific progress, they seemed to describe phenomena of different kinds . Yet, some relationships, discovered earlier by the likes of gay - lussac, avogadro, and boyle, consistently crossed that divide, such as the proportionality of gas pressure (a mechanical attribute) and temperature (a thermodynamic one) in a container of fixed volume . Moreover, simple observations remained challenging to reconcile on both sides of the divide, such as why matter can apparently be heated up to arbitrarily high temperatures but not cooled off below a certain limit (absolute zero or 273.15c) and the continuously jittery (brownian) motion of particles visualized under a microscope . Between 1856 and 1871, kroenig, clausius, maxwell, and boltzmann developed a kinetic theory explaining thermodynamics in terms of mechanics, based on pioneering ideas originally proposed more than a century earlier . Specifically, temperature relates to the average quadratic velocity of a large number of microscopic particles and pressure to their momentum . These relationships intuitively match the observation that, when warming up one's hands by rubbing them together, the faster the movement, the higher the generated heat . Entropy is related to the number of possible states a system can be in, which clarifies why disorder tends to increase in the absence of other constraints . The demonstration of the equivalence between thermodynamics and mechanics is as convincing and direct as the derivation of the corresponding thermodynamic and mechanical laws . In particular, furthermore, this link solved the mystery of the lower bound of temperature: there is no practical limit to the maximum speed that particles can have, but they cannot go any slower than being perfectly still (corresponding to absolute zero). Notably, the kinetic theory also led to the famous 1905 einsteinian explanation of brownian motion (recounted in). Another striking example of scientific advancement linking phenomena of different kinds is the explanation of optics in terms of electromagnetism . The phenomena of refraction and diffraction are fully explained by maxwell's equations of electrodynamics once light is understood as electromagnetic wave . Yet the properties of mirrors and the passage of light through various media (such as air and water) seem so different from the phenomena of electric current and magnetic dipoles . Other illustrations of the same relationships are the quantum physics foundation of chemistry, the genomics bases of genetics, and the explanation of neuronal firing in terms of voltage - dependent sodium and potassium channels . When mendeleev compiled the first draft of the periodic table of the elements, there was no physical justification of the observed proportions of chemical reactions: the truth and correctness of the table relied on its predictive power and descriptive elegance . Mendeleev's few initial mistakes, most of which he himself corrected in subsequent versions, were due to using the atomic mass (the observed chemical weight of a substance) rather than the atomic number (the number of protons in the nucleus) as the organizing principle . The atomic nucleus was discovered 42 years later, just 4 years after mendeleev's death . Thus his corrections, required to describe then - available data parsimoniously, de facto predicted the atomic number nearly half a century prior to its actual discovery . The subsequent development of quantum mechanics showed that the atomic number emerged as a mathematical necessity from more fundamental assumptions, such as schrdinger's wave equation or heisenberg's uncertainty principle . Today we readily accept that many chemical phenomena (e.g., the tendency of metals and halogens to combine, as well as their valence ratios) are ultimately explained by quantum mechanics, even if these two phenomena would appear to relate to different and independent aspects of reality . We purport that the illusion of mystery at one level (e.g., the law of gases, light diffraction, and the combining ratios of chemical elements) evaporated as soon as those same sets of properties were discovered or demonstrated at a different level (particle kinematics, electromagnetic waves, and quantum orbitals). Breakthroughs of these sorts are fundamentally distinguishable from hand - waving just - so speculations because once the parallel is drawn, it systematically explains and predicts an entire body of observations rather than isolated phenomena . Here we surmise that the content and meaning of mental states, the most inescapable yet ineffable puzzle of human cognition, will eventually be understood as a direct reflection, if not simply an aspect, of brain computation, much like thermodynamics is statistical mechanics . Given the complexity of brain dynamics and of our inner experience, we expect the mathematical constructs connecting neuroscience and mind to be far less simple than newton's, maxwell's, or schrdinger's equations . The principle of what would count as an explanation, however, remains the same: to solve the mind - brain conundrum one only needs to show that the math of the mind and the math of the brain are equivalent . We are not simply proposing mental properties to be probabilistically supervenient on brain properties, that is, that they can be inferred statistically from brain measures within any given error rate . On the contrary, we are asserting the possibility of a formal equivalence between the two, through all temporal scales and plastic changes . The explanatory power of mathematical theory in neuroscience is recognized in principle, but the extent of its reach has not yet been fully realized, and the path forward has never been chartered before . This is in stark contrast to the third simulation - based leg of scientific progress (complementary to experiments and theory), which is blossoming into maturity in computational neuroscience and cognitive modeling alike and in the study of consciousness in particular [51, 52]. To explain the equivalence of brain and mind by mapping them onto each other, it is essential to identify the relevant levels of analysis in order to define proper mathematical formalisms for their quantitative description . We start from the brain in this section and tackle the mind in the next . Specifically, how neurons are connected to each other constrains network dynamics and therefore determines the possible flow of information transmission . A human brain has an estimated ~10 neurons and 10 synapses, which could in theory form a humongous number (~10 while neuroanatomy provides the foundational roadmap of information transmission in nervous systems, neural activity is itself characterized by chaotic dynamics typical of complex systems [59, 60]. As these aspects are particularly relevant to conscious brain function, a full understanding of the brain as it relates to mental content will have to integrate adequate accounts of both neural dynamics and connectivity [6264]. Nevertheless, the network architecture specification is absolutely central to the assumed correspondence between spatial - temporal patterns of neural spiking and mental states . The notion of connectomics, characterizing the circuit blueprint of the nervous system, has progressively grown close to practical feasibility with the recent dramatic advancements in genetic manipulations allowing for multicolor microscopic visualization [66, 67]. The former is further distinguished in the dense reconstruction of the entire synaptic matrix and the statistical potential of synaptic connectivity, both highly relevant to computational processing [7173]. In contrast, the much coarser description of regional connectivity has less direct implications for a mechanistic understanding of brain cognition . However, this latter approach is also substantially more realistic to achieve in the near future, using existing histological techniques in animal models [74, 75] or noninvasive imaging in humans [76, 77]. A number of big - science as well as grass - root data acquisition efforts are underway in both cellular and regional connectomics . These include the human connectome project [78, 79], the 1000 connectomes project [80, 81], the mouse brain architecture project, and the flycircuit database, among many others . This flurry of developments along with nonconventional approaches (e.g.,) is generating a wave of optimistic expectation in the research community that massive neural connectivity data will become increasingly available in the foreseeable future . The branch of mathematics dealing with connectivity is graph theory . In light of the previous considerations, it is not surprising that graph theory has become a considerably popular topic in neuroscience (e.g., [85, 86]). It is remarkable that important properties of general graphs that have been found to apply to many types of networks, including random connections, small - world attributes, scale invariance, and motif distributions, are prominently relevant to neural circuits [9194]. The application of graph theoretic analysis to neural circuit has already revealed a number of features, including network communities and rich clubs, but also general principles of wiring economy and network organization as well as potential implications of circuit structure on signal communication [99, 100]. It is important to stress that, while two cells are never exactly alike, neurons can be organized in distinct classes such that neurons within each class are much more similar to each other than across classes . Thus the statistical properties of brain connectivity are likely to be strongly determined at the level of connection probability among neuron classes . Initial progress is being made in the application of the relevant field of mathematics, stochastic block modeling, to this problem [102, 103]. Two further facets are worth considering in the characterization of the brain in terms of its network connectivity . While in invertebrates it is sometimes possible to recognize the same individual neurons across subjects, in mammals it is not even possible to match the same types of neurons bilaterally within subject, such as in motor neurons innervating symmetric muscles . In humans, intersubject variability is already very considerable at the regional level and can be expected to be extraordinary large at the level of individual neurons across subjects . The second vital element of brain circuitry is structural plasticity, that is, dynamical changes in the synaptic connectivity not just during development but throughout adulthood . In the cortex of normally behaving mice, for example, 4% of axonal boutons change over the course of a few months, with similar proportions reported in dendritic spines . Abundant experimental evidence suggests that this form of plasticity is activity- and experience - dependent [108111]. This is just one of many mechanisms underlying neural plasticity across spatial and temporal scales, from short- or long - term alterations in synaptic strengths to neurogenesis, which are believed to support memory storage [113, 114]. Much as the brain is in constant flux and its functional connectivity continuously changes with every spike and synaptic discharge, so is the mind never exactly the same before and after instantiating each and every subjective representation . These aspects of both brains and minds are strongly resonant with heraclitus' panta rhei . One may find it disturbing that despite knowing so much about the structure and activity of the brain, we have a hard time guessing but it is somehow even more peculiar that in spite of direct, detailed, continuous, and complete access to each and all conscious mental states we experience, we find it difficult to describe them comprehensively, let alone quantitatively . Indeed, it seems absurd that we can measure the concentration of substance p in single neurons to the fifth significant digit; yet we can only measure the resulting sensation of pain semiqualitatively on a 7-point discrete scale . In order to bring the study of conscious content into the realm of hard science, we need to devise a quantitative measurement system for subjective states . Language has often been considered a convenient proxy to access mental states, if not the most direct tool to describe them . The scientific characterization of the meaning of language, or semantic analysis, has a long history and remains one of the most active research areas in (computational) linguistics . Here we do not aim to review or even to provide a balanced commentary on the state of the art of semantic analysis techniques . Instead, we introduce and explain a very specific, nonconventional approach to this problem that is particularly pertinent to the topic of this spotlight paper . Most if not all of the best known computational methods of semantic analysis are based on (variations of) the common principle that the meaning of words relates to the contextual occurrence of their use in language . For example apples, oranges, and grapes tend to be used in similar contexts as reflected by their cooccurrence with similar words in the same sentence (e.g., eat, ripe, juice, and vitamin). Thus, they share similar semantics (they are all types of fruit). The notion that word meaning relates to the (relative) frequency of their cooccurrence is shared by many broadly adopted approaches, including latent semantic indexing, latent dirichlet allocation, hyperspace analogue to language, and many others . In practice, these techniques rely on the identification of statistical patterns of word usage in large - scale text corpora by computational parameter extraction . Although the details vary among types of computational semantic analysis, words (or more generally, concepts) are often allocated to a multidimensional abstract space such that the location of each concept reflects its meaning . These spaces are sometime referred to as semantic maps . Meaningful semantic dimensions of these spaces can be associated with their geometrical shape, a bit like the location on a globe can be described with the polar and azimuth angles, or the size of a cigar can be described by its length and thickness . For instance, all fruit words in the previous example would be located in the same region of the space . By nature of its own principle, latent semantic analysis and its variations generate results that are highly context dependent . In other words, the semantics extracted from a cookbook are typically quite different from those detected in movie reviews or obituaries . In fact, use of nonhomogeneous collections of corpora from different domains typically fails to yield meaningful semantics . Moreover, this general class of methodologies tends to produce a large number of highly specific dimensions . A rather complementary (and historically precedent) goal of lexical semantics has been to seek the fundamental (or at least context independent) dimensions of word meaning . Perhaps the most seminal study in this sense has been that of osgood's semantic differentials . In that work, subjects were asked to rate a large number of words in various hand - picked dimensions defined by two opposite extremes (e.g., soft - hard, fast - slow, clean - dirty, valuable - worthless, and fair - unfair), using a likert (discrete) scoring scale . Subsequent analysis identified three principal dimensions that were robust to cultural and geographical differences, namely, evaluation (also known as valence: good - bad), potency (strong - weak), and agency (active - passive). A limitation of these studies and other similar psychometric approaches is that they involve human subjects and arbitrary choices of starting terms . Word meaning has of course also been characterized for thousands of years in many languages and cultures through the creation of dictionaries . Here the beginning of modern times can be considered to correspond to the systematic (but again, ultimately arbitrary) classification introduced by roget's thesaurus of english words and phrases, now accessible online 160 years after its original publication . Among contemporary efforts, the most comprehensive academic resource is princeton's wordnet . Yet, it remains to be established if and how formal ontological theories could map semantic spaces such as those generated by latent semantic analyses . We have recently introduced a novel paradigm for semantic mapping that allows systematic construction of a low - dimensional metric system for the context - independent (fundamental) meaning of words . Our approach combines certain elements of osgood's work (use of antithetical meanings) with the scalable use of objective dictionaries . Specifically, using a novel self - organization process, we constructed a semantic map of natural language that simultaneously represents synonymy and antonymy . Synonyms and we extracted these relationships from digitally accessible dictionaries (microsoft word and princeton's wordnet) in each of several languages (english, french, german, and spanish). For each dictionary and language, we initially allocated words at random locations in a finite, multidimensional spherical space . Then we started moving the position of every word following a simple rule: every word would attract its synonyms and repel its antonyms . Thus, pairs of synonyms would tend to move closer to each other, and pairs of antonyms would move farther apart (within the bounds of the multidimensional sphere). This process converges in the sense that all words reach a stable position that could not be further improved in terms of proximity to synonyms and distance from antonyms . The resulting space only had a limited number (~4) of statistically significant dimensions . This means that, even if the starting space is a homogeneous sphere of many (~100) dimensions, the resulting emergent shape can be quite completely described with just four numbers . Most importantly, the emergent semantics of the map's principal components are clearly identifiable: the first three correspond to the meanings of good / bad (valence), calm / excited (arousal), and open / closed (freedom), respectively . The semantic map is sufficiently robust to allow the automated extraction of synonyms and antonyms not originally present in the dictionaries used to construct the map, as well as to predict connotation from their coordinates . The map's geometric characteristics include a bimodal distribution of the first component, increasing kurtosis of subsequent (unimodal) components, and a u - shaped maximum - spread planar projection . Both the semantic content and the main geometric features of the map are consistent between dictionaries, among tested western languages, and with previously established psychometric measures . Some of the mathematical formalism and speculative interpretations are elaborated in a second follow - up paper . Interestingly, the main emerging dimensions of this semantic map loosely correspond to the primary modulatory neurotransmitter systems in the mammalian brain . The previous paradigm can be expanded with appropriate adaptations to extract additional, independent dimensions of word meaning by considering other linguistic relations besides synonyms and antonyms . In particular, (hypernyms and hyponyms) captures the abstractness (or more precisely the ontological generality) of words . For example, the statements mickey is a mouse, the mouse is a rodent, and a rodent is an animal reflect a hierarchy of concepts from the more concrete, or rather specific (mickey), to the more abstract / general (animal), whereas rodent is a hypernym of mouse and a hyponym of animal . In this paper might be confused to indicate how common a term is (usage frequency). Is - a relationships are commonly used in dictionary definitions following the classic recipe of aristotle's logic a is a type of b with property c . However, hypernyms and hyponyms are seldom listed in immediately machine - readable form in digital collections, the way synonyms and antonyms are . One exception is provided once again by wordnet, which explicitly provides is - a relationships among many of its terms . Unlike synonyms and antonyms, which are symmetric relations (if a is synonym of b, b is synonym of a), hypernyms and hyponyms are directional and mutually antisymmetric (if a is hypernym of b, b is hyponym of a). We thus changed the form of the energy functional in the previously described optimization procedure . The resulting allocation of words in space yielded a ranking of all terms along a single dimension, that is, a simple scalar measure of their abstractness (ontological generality). For example, the ten top scoring terms (out of ~124,000) are entity, physical entity, psychological feature, auditory communication, unmake, cognition, knowledge, noesis, natural phenomenon, and ability . The bottom (11 ex aequo) of the list reads edmontonia, coelophysis, deinocheirus, struthiomimus, deinonychus, dromaeosaur, mononykus olecranus, oviraptorid, superslasher, utahraptor, and velociraptor . One advantage of a metric system is that words can be compared in terms of their abstractness even if they are unrelated, for example, the term governance (whose abstractness value is 1.726) can be determined to be more abstract than newspaper (abstractness of 0.541) even if there are no (sequences of) is - a relationships connecting them . Moreover, because the measure is quantitative, it allows evaluation of relative comparisons . For instance, power (1.805) is more abstract than revolutionary (1.070). However, the generality of governance relative to newspaper (abstractness difference: 1.726 0.541 = 1.185, abstractness ratio: 1.726/0.541 = 3.190) is greater compared to that of power relative to revolutionary (abstractness difference: 1.805 1.070 = 0.735, abstractness ratio: 1.805/1.070 = 1.687). This opens the possibility to establish a probabilistic estimate of whether a word is more abstract than another . The metrics of context - independent word meaning along the principal dimensions described previously can be applied to characterize declarative mental states . The most straightforward application is to quantify the content of verbal examples along the main dimensions of the map . It should be noted that the semantic map described here represents a complementary, rather than alternative, tool to more established latent semantic analyses . While maps produced by the latter are corpus (and context) dependent, this space adds general dimensions that are applicable to all corpora and context . For example, we successfully used the map to rank online collections of movie reviews and biomedical abstract based on the average measure of their words (the very primitive bag of words approach) along our dimensions . We indeed found that the first dimension (good - bad) was an excellent quantitative predictor not only of the movie critique score but also (together with the second dimension) of its genre (high valence and arousal: action, low valence and high arousal: drama, high valence and low arousal: romantic comedy, and low valence and arousal: documentaries). It is also possible to extend the same approach to the has - a relationship (holonyms / meronyms), which is also explicitly included in wordnet . For example, in the sentence a mouse has a whisker, the mouse is holonym of whisker and whisker is meronym of mouse . This relationship is (like is - a) also antisymmetric, and we thus expect to be able to extract a semantic mapping provides a possible approach to quantifying mental states that can be expressed declaratively . Mental states, or more practically words, sentences, paragraphs, and text in general are allocated in a multidimensional space such that their distances and relative positions reflect particular semantic relationships, including hypernymy / hyponymy (is - a), holonymy / meronymy (has - a), synonymy / antonymy (is similar / opposite to), and in principle many others, such as causation and cooccurrence . In this framework, mental states and their relationships if one believes that (at least some) mental states reflect properties of outer reality, it is possible to conceive reality itself as occurring in a giant graph in which any possible observable is a node, and edges correspond to probabilities that two observables would cooccur . We call this conceptual construct the universal reality graph . In this view, reality would unfold in time as a sequence of events constituting patterns of activation of subsets of nodes and all edges among them within the university reality graph . Any agent capable of observation will witness a subset of these activation patterns, that is, a sequence of partial events, each consisting of a collection of active nodes and edges . At each moment of experience, every agent would learn some (but not all) of those associations s / he witnesses . In this process, the agent would progressively form a mental graph representing part of his / her experienced history, which is itself part of the general occurrence of reality, sampled at each instant from the universal reality graph . While this notion of reality as a graph is purely speculative, even more extreme theories have proposed that physical reality is ultimately a product of information (it from bit), rather than the other way around . Most importantly, the possibility to conceive reality as a graph offers interesting vistas on the solution of the mind - brain problem . If agents form graph - like minds to represent (and therefore predict) their experience of graph - like reality, it stands to reason that the fittest physical substrates selected by evolution to encode these representations be themselves graph like, namely, brain networks . The relationship between minds and brains could then be resolved as a mapping between their respective graphs and their embeddings . In this framework, the fundamental operation to grow a mind is pairwise association between observables, that is, establishment of edges between nodes in the mental graph based on corresponding experiences in the reality graph . An interesting aspect of mental representation is that we only learn a small fraction of associations from those observed in reality . In particular, we have recently proposed that this constraint may be a consequence of the spatial relationship among the tree - like shaped neuronal axons and dendrites that underlie brain connectivity [135, 136]. Specifically, in order for new synapses to be formed, the axon of the presynaptic neuron must be sufficiently close to a dendrite of the postsynaptic neuron, arguably because of preexisting connectivity with other neurons encoding for related knowledge . These ideas are also consonant with the information integration theory (iit) of consciousness, which is emerging as a leading candidate among the fundamental theories of mental content . The underlying assumption of iit is that consciousness is fundamentally a property of information processing . Specifically, according to the iit, when a brain (or in principle any other computing device) is in a particular state, its amount of consciousness, called phi, depends not only on the actual content represented in that state but also on the absence of all contents represented in the states that are not being (but could be) instantiated . Thus silent neurons contribute to the conscious state as much as the active neurons, because consciousness depends as much on the content that could be represented by the network as on the content that is actually being represented . Therefore, consciousness is a product of the integrated activity in the network and is measured by information integration, a property that has been defined in graph structures . While the iit profoundly links consciousness to information, its cognitive underpinning is shared by other theories (e.g.,) and experimental approaches . A crucial and unique outcome of iit, however, is that the definition of integrated information enables a geometric characterization of mental states or qualia . This can in principle provide a neurally based bottom - up correlate to the spaces that emerge from top - down semantic mapping of natural language . If the information processing product of neural network activity can be shown to correspond mathematically to a quantitative description of subjective mental content, the brain - mind problem would be effectively resolved . Information is not only an essential element of consciousness, reality, and brain activity, but also of communication among conscious agents . More precisely, what does it mean to the speaking individual, and what does it mean to the listener? Shannon's notion of information captures the reduction of uncertainty of the listener's mind upon receiving the message . Assuming that the second individual had no idea of what time it was, whether the first person was rested or fatigued, and so forth, communication is indeed informative . Mapping brain and mental states, however, opens another perspective on the meaning of communication . Consider the brain / mind state x of the first individual (being tired, etc . ), and suppose that the goal of the message was to instantiate x in the second individual . As a result of communication, however, the second individual's brain / mental state is y rather than x. if y equaled x, communication would be 100% perfect, but that is never the case . It's almost midnight may be fairly close to the speaker's meaning of those words, but even in that case, midnight could be associated with different feelings and memories in the two individuals, and the term almost might be interpreted as 20 minutes by the first individual and as 2 minutes by the second . When we analyze the second portion of the message, i'm tired, we realize that the listener will think of his / her notion of tiredness, which is at best a coarse approximation of the actual state expressed by the speaker, for example, in terms of physical versus mental, chronic versus acute, concerned versus conversational, and so forth . The connotation of i had such a day is even more prone to subjective interpretation . The listener may think of his / her days s / he would describe as such a day, but those days and associated sentiments are likely quite different from the events and related mental state the speaker was referring to . How much of the intended meaning is effectively transmitted between communicating conscious individuals on average? Even in the most favorable cases of colleagues discussing their joint work or spouses talking about their family, we speculate that communication hovers between 50% and 85% . If this is the case, the mean human communication between any two individuals (including casual interactions) might be 10% or less . Even reminiscence and planning (retrieval of autobiographic and prospective memories, resp . ), as well as other forms of internal dialogue, can be viewed as special cases of communication (with oneself). Such a type of communication between two instances of the same individual at different points in time can be expected to be much more effective than between different human beings, but even in those situations it will not be perfectly effective, as the mind is in constant flux . In all cases, mental state quantification by semantic mapping and its corresponding neural correlates in brain activity spaces could dramatically enhance communication effectiveness, deeply altering human relationship . The relationship between mind and matter has perhaps been, in one form or another, the most debated issue in the history of human thought, and it still constitutes, in the modern mind - brain incarnation, an open scientific and philosophical problem . Specifically, why do certain brain states feel like something, and why specific brain states feel the way they do? We have proposed that a satisfactory answer can ultimately come from mathematics, if the abstract spaces of brain activity and mental content can be quantitatively characterized and geometrically mapped onto each other . Such a solution will connect the conscious mind and the relevant aspect of brain states by demonstrating the equivalence of their properties, much like statistical mechanics and thermodynamics are nowadays accepted as one and the same phenomenon, even if they are practically treated as distinct for every day purposes . We argued that semantic maps constitute a useful initial framework to establish a rigorous description of the mind and that network connectivity provides the most informative constraint on brain dynamics . However, defining the proper mathematical states to effectively bridge brain and mind still constitutes a formidable challenge . State - of - the - art semantic maps only scratch the surface of the necessary quantification of the human mind . Next - generation voice recognition and optical character recognition software programs might soon enable real - time acquisition and analysis of the complete life - long natural language corpus experienced by an individual . Exhaustive compendia of semantic relationships could be extracted from such a resource, enabling the creation of a comprehensive semantic map for that individual . While the main obstacle in quantifying mental content appears to be the required paradigm shift towards a science of first - person perspective, neuroscience faces mostly a technological hurdle in creating brain - wide neuron - level maps of network connectivity and activity . Specifically, existing techniques can indeed map all of synaptic circuitries, but only in a very small volume (a fraction of cubic millimeter) of nervous tissue, only in animal models, and not in vivo . Other techniques to analyze neuronal anatomy, only hinting at the potential connectivity [75, 145], are possibly scalable to entire brains of live animals [146, 147], but again not human beings, let alone in normal behavioral conditions . The only noninvasive imaging techniques available to investigate the human brain (e.g.,) are by many orders of magnitude too coarse to probe the level of neurons and spike . Scenarios have been proposed to solve the technological gap between small - scale animal - model neuron - level analysis and a full activity map of all neurons and synapses of a sentient human brain, such as the eventual adoption of nanotechnology . One present - day partial solution is to use molecular homology to identify existing correspondences between neuron types in rodents and humans by comprehensive genetic mapping and single - neuron sequencing . The subsequent extension of rodent brain connectivity to human cognitive architecture would only be tentative, requiring extensive computational testing and refinement by multiscale simulation . An initial pilot project in this regard might tackle a suitable brain region (and related computational functions), such as the mammalian hippocampus . Assuming that, at least in principle, technological advancements enabled accumulation of sufficient datasets to adequately map the neuronal activity of the human brain, such a feat would likely involve massive automation . High - throughput, machine - acquired, and large - scale data poses the outstanding matter of human interpretation [155157]. This issue has recently promoted considerable growth in the field of neuroinformatics, that is, the establishment of an information framework for neuroscience (e.g., databases and other electronic resources), which is especially needed in computational neuroanatomy . Recent initiatives have proposed a formalism to represent connectivity structure in neuronal network models and seeded web - based multimodal connectivity databases . A parallel informatics effort is required to enable storage, manipulation, and analysis of machine- and human - readable empirical data on cognitive functions, behaviors, and introspection [162, 163]. The proposed vision offers a path towards a mathematical solution of the relationship between brain and mind that is consistent with contemporary philosophical positions . Tremendous advancements in physics, chemistry, and biology provided an increasingly unified understanding of the material world . Bridging neuroscience with a quantitative description of inner subjective life may provide a fundamental closure to human scientific inquiry.
A human being exposes himself to a variety of injuries caused by numerous forces like vehicular accident, social conflict, terrorism, crimes, wars, industrial accident and fall from a height . The commonly injured organs are the liver, spleen, kidney, intestines, stomach, pancreas, urinary bladder and vessels . Previously all patients with bta ended up in laparotomy and were managed according to the organ injuries . The advent and development of new techniques like laparoscopy minimally invasive surgery (mis) have a diagnostic as well as definitive therapeutic role in bta . The availability of sophisticated instruments, equipments and expert anaesthesiologists make laparoscopy an attractive technique for diagnostic and therapeutic measures in bta . Haemoperitoneum with stable vitals and injury to the liver, spleen, bowel, mesentery and bladder can be managed by laparoscopy . Advanced laparoscopic techniques include bowel resection and anastomosis, ligation of blood vessels can be as efficiently utilised in bta as an elective open surgery. [35] one can visualise the peritoneal cavity and act expeditiously if needed (i.e., laparotomy, laparoscopic - assisted intervention, or only observation) at the time of laparoscopy . This study was planned with the objective of evaluating the therapeutic efficacy of laparoscopy in managing the organ damage in bta . After institutional ethical committee approval and informed written consent from the patients, a prospective randomised clinical study was carried out in 25 adult patients of either sex, scheduled for laparoscopic intervention in the surgical ward of the government medical college and sir t. hospital, bhavnagar . Patients who sustained haemoperitoneum, confirmed in ultrasonography (usg) or computed tomogram (ct scan), with relatively stable haemodynamics, were enrolled in this study (blood pressure> 80 mm of mercury systolic and> 15 ml / hour urine output). Patients with unstable haemodynamics, even after three units of whole blood transfusion (1 unit = 300 cc) and associated edh (extradural haemorrhage) / sdh (subdural haemorrhage), compound fracture, spine fracture, severe chest injury with low spo2 (<90%), haemodynamically unstable, anticipated difficult endotracheal intubation and pregnancy, were excluded . The three main ports are: umbilical port (10 mm)right - sided port (5 mm / 10 mm) lumbar at anterior axillary lineleft - sided port (5mm/10 mm) lumbar at anterior axillary lineextra port, if required, is made according to the organ injury usually in the subxiphoid region or in the lower abdomen . Umbilical port (10 mm) right - sided port (5 mm / 10 mm) lumbar at anterior axillary line left - sided port (5mm/10 mm) lumbar at anterior axillary line extra port, if required, is made according to the organ injury usually in the subxiphoid region or in the lower abdomen . If no complex injuries are found and the patient is haemodynamically stable, focussed therapeutic laparoscopic intervention is done, such as, primary closure of the bowel perforation, primary repair of the bladder rupture in two layers, electrocauterization and spraying of feracryline (hemolock) solution at the injury site on the liver and spleen (contusion, laceration or tear), with no active bleeding, without disturbing the preformed hematoma. [81114] the three main ports are: umbilical port (10 mm)right - sided port (5 mm / 10 mm) lumbar at anterior axillary lineleft - sided port (5mm/10 mm) lumbar at anterior axillary lineextra port, if required, is made according to the organ injury usually in the subxiphoid region or in the lower abdomen . Umbilical port (10 mm) right - sided port (5 mm / 10 mm) lumbar at anterior axillary line left - sided port (5mm/10 mm) lumbar at anterior axillary line extra port, if required, is made according to the organ injury usually in the subxiphoid region or in the lower abdomen . If no complex injuries are found and the patient is haemodynamically stable, focussed therapeutic laparoscopic intervention is done, such as, primary closure of the bowel perforation, primary repair of the bladder rupture in two layers, electrocauterization and spraying of feracryline (hemolock) solution at the injury site on the liver and spleen (contusion, laceration or tear), with no active bleeding, without disturbing the preformed hematoma. [81114] demographic profile of patients laparoscopic intervention required in different patients laparoscopic intervention required in blunt trauma abdomen data suggested that two - thirds of the cases are due to road traffic accidents [figures 15]. Splenic laceration near hilum (grade-2 injury) laparoscopic urinary bladder repair post laparoscopy scar on discharge drain placement in the right sub - diaphragmatic space in a liver injury in the present study a total of 25 cases of bta were studied, out of which 24 cases (96%) were managed laparoscopically and one was converted into an open mini - laparotomy . Graph 1 suggests different organ injuries in bta, liver being the most common in our study, in contrast to western countries where the spleen is the most common . No injuries were detected in two patients other than the non - expanding retroperitoneal haematomas which were left untouched . Hence, the diagnostic value of laparoscopy (98%) has a tremendous value, which corresponds to the hamish foster series (89%) with zero% negative laparotomy . With 04% failure rates, a single laparoscopy is converted into a mini - laparotomy, for large ileal perforation and hematoma, and managed by resection and anastomosis with the help of a small umbilical port cosmetic incision . Another single patient, who was referred to a higher centre of management of pulmonary embolism on the first post - operative day, not maintaining spo2 even with 6 l of o2 support, due to pulmonary embolism, was not included in the study . Table 4 reveals the post - operative management in patients of bta treated by laparoscopy and supports the conclusion . Post - operative management practically no complication was found related to laparoscopy in present study . In second and third decade of life the liver and spleen are common organs involved in bta . With the advent of the current laparoscopy laparoscopy provides early mobilisation, early oral intake, fast recovery, early resumption of work, reduced post - operative stay in the hospital and analgesic requirement, with early discharge as compared to laparotomy . Limitations: it requires sophisticated instruments and general anaesthesia; hemodynamic stability of the patient, poor visualisation of the seventh and eighth segments of the liver, posterior surface spleen, retroperitoneal organ, pancreas and second part of duodenum . The question addressed by this study is whether introduction of an aggressive laparoscopy programme would find its acceptance, and will it make a difference or not? Our data clearly shows that this indeed will occur, however, it requires a further, prolonged, prospective study for obtaining an even better conclusion and interpretation . With advancement in equipment, more people getting trained and the surgeon's being able to perform technically difficult manoeuvres laparoscopically, it appears that laparoscopy is now closer to replacing emergency laparotomy in the forthcoming future, for the management of blunt abdominal injury.
For example, storm types have been defined based on the magnitude of the dst index: minor storm (30 nt> dst> 50 nt), moderate storm (50 nt> dst> 100 nt), intense storm (dst <100 nt), and great storm (dst <250 nt) [e.g., gonzalez et al ., 1999]. The dst index is designed to be a measure of the magnetic perturbation near the equator from currents flowing above the ionosphere . As such, it is adjusted for the sq dayside ionospheric current system and for the secular variation due to changes in the internal magnetic field of the earth . The kyoto dst index baseline is corrected for the secular variation of the earth's internal magnetic field by making a quadratic fit to each of the four dst observatories by using the last 5 years of magnetic data from that observatory . Each year's contribution to the quadratic fit is based on the five quietest days of each month of that year . (the five quietest days of each month produce sixty values for that year, which are then averaged to produce a single value for that year . An additional point is added whose value is the baseline value at the end of the current year as determined in the first step, and then a second quadratic fit is done using this additional point [sugiura and kamei, 1991]). Is determined using a different set of years, a discontinuity occurs at the end of each year and because quiet days during solar maximum may not be as quiet as quiet days during solar minimum, the average dst value may underestimate the change in magnetospheric activity between solar maximum and solar minimum . The main point of this report is that this is the case: the kyoto dst index underestimates the solar cycle variation of magnetic activity . We have previously developed a model of the kyoto dst index based solely on the solar wind [temerin and li, 2002, 2006]. This model does a good job of duplicating the kyoto dst index for the years 19952002 using the solar wind magnetic field, velocity, and density as input . The model also had a baseline correction with a single discontinuity at the end of 1999 as we were not then aware that there was in fact a discontinuity at the end of each year in the kyoto dst index . We have now extended the model to years 19952009 using omni solar wind data (instead of using solely wind and ace satellite data), added effects based on the f 10.7 index and effects using all three components of the solar wind velocity instead of just the vx component . We report on the effect of changing the baseline correction to the model . To find the baseline correction in the model we have made a quadratic fit to each year separately . The coefficients of each year's quadratic fit are found by minimizing the rms error between the model and the final kyoto dst index . The resulting baseline correction in our model the solar cycle has a minimum based on the sunspot number and the f 10.7 index in 1996 and again in 2008 and 2009 and a maximum in 2000 and 2001 . Geomagnetic activity has a double peak in 2000 and an even larger peak during the declining phase of the solar cycle in 2003 . The three coefficients in each quadratic expression are found by minimizing the rms difference between the model and kyoto dst index . Figure 1 shows the baseline correction added to the model to minimize the rms difference between the model and the kyoto dst index . In particular, conversely, the values in figure 1 can be regarded as the values that need to be subtracted from the kyoto dst index to make it agree with the model . Figure 2 shows that the model with the baseline correction agrees well with the kyoto dst index . A comparison of the model output with the kyoto dst index for 3.5 months around the end of the year 2002 . The point of the figure is to show the good agreement between the model and the kyoto dst index . Figure 3 (top) shows the model error without the baseline correction for the same time period . Figure 3 (bottom) shows the model error after the baseline correction is applied . (the kyoto dst index itself changes from 16 nt from the last hour of 2002 to 4 nt for the first hour of 2003 .) (top) the difference (i.e., the error) between the model and the kyoto dst index when the baseline correction is not applied to the model for the period shown in figure 2 . The point of the figure is to demonstrate that the kyoto dst index can have significant discontinuities . Figure 4 (top) shows the 27 day running average of the kyoto dst index and of the model if no baseline correction is applied . Note that there is less solar cycle variation in the averaged kyoto dst index than in the modeled dst index . There is substantially more solar cycle variation in the modeled dst index when no baseline correction is applied . Since the kyoto dst index is corrected using quiet days and since quiet days during solar maximum are likely to be less quiet than during solar minimum, the baseline correction we apply to our model to make it agree with the kyoto dst index removes much of the solar cycle variation in the averaged data . The modeled dst with no baseline correction, however, does not remove the solar cycle variation in the process of removing the secular variation . (top) a comparison of the 27 day running average of the kyoto dst index with the model with no baseline correction applied . (bottom) the good agreement between the 27 day running averages when the baseline correction is applied to the model . It is important to note that our point is that the model without the baseline correction is a far more accurate representation of the effects of magnetospheric currents that affect the magnetic field at dst observatories than the kyoto dst index . Figure 4 (bottom) compares the 27 day moving averages once the baseline correction is applied to the model . Now the agreement between the model and the kyoto dst index is very good (correlation coefficient = 0.981). (except for the baseline coefficients, the coefficients of the model were optimized using only data from the years 19952002 .) In addition, we have looked at all the year end final kyoto dst data between 1957 and 2009 and found that the average change at the end of the year (5.9 nt) is more than twice as large as in each of the 2 h before and after (2.5 nt) the end of the year, implying an average discontinuity in the kyoto dst index of between 5 and 6 nt at the end of each year . The largest discontinuity appears to have occurred at the end of 1982 when the kyoto dst index decreased by 20 nt for no apparent reason . For any individual year it is not possible to determine the discontinuity by the change at year's end since some of the change can be physical . But by comparing the model with the dst index as in figure 3, it can be estimated well . We conclude that the process of removing the secular variation from the kyoto dst index inadvertently removes some of the solar cycle variation . Thus, the average value of the kyoto dst index underestimates the modulation effects of the solar cycle on magnetospheric activity and in particular on the ring current . Conversely, if one uses the kyoto dst index to remove the magnetospheric current contribution to the magnetic field to determine the secular variation of the magnetic field, one will also get an incorrect result for the secular variation . To find the baseline correction in the model we have made a quadratic fit to each year separately . The coefficients of each year's quadratic fit are found by minimizing the rms error between the model and the final kyoto dst index . The resulting baseline correction in our model the solar cycle has a minimum based on the sunspot number and the f 10.7 index in 1996 and again in 2008 and 2009 and a maximum in 2000 and 2001 . Geomagnetic activity has a double peak in 2000 and an even larger peak during the declining phase of the solar cycle in 2003 . The three coefficients in each quadratic expression are found by minimizing the rms difference between the model and kyoto dst index . Figure 1 shows the baseline correction added to the model to minimize the rms difference between the model and the kyoto dst index . In particular, conversely, the values in figure 1 can be regarded as the values that need to be subtracted from the kyoto dst index to make it agree with the model . Figure 2 shows that the model with the baseline correction agrees well with the kyoto dst index . A comparison of the model output with the kyoto dst index for 3.5 months around the end of the year 2002 . The point of the figure is to show the good agreement between the model and the kyoto dst index . Figure 3 (top) shows the model error without the baseline correction for the same time period . Figure 3 (bottom) shows the model error after the baseline correction is applied . (the kyoto dst index itself changes from 16 nt from the last hour of 2002 to 4 nt for the first hour of 2003 .) (top) the difference (i.e., the error) between the model and the kyoto dst index when the baseline correction is not applied to the model for the period shown in figure 2 . The point of the figure is to demonstrate that the kyoto dst index can have significant discontinuities . Figure 4 (top) shows the 27 day running average of the kyoto dst index and of the model if no baseline correction is applied . Note that there is less solar cycle variation in the averaged kyoto dst index than in the modeled dst index . There is substantially more solar cycle variation in the modeled dst index when no baseline correction is applied . Since the kyoto dst index is corrected using quiet days and since quiet days during solar maximum are likely to be less quiet than during solar minimum, the baseline correction we apply to our model to make it agree with the kyoto dst index removes much of the solar cycle variation in the averaged data . The modeled dst with no baseline correction, however, does not remove the solar cycle variation in the process of removing the secular variation . (top) a comparison of the 27 day running average of the kyoto dst index with the model with no baseline correction applied . (bottom) the good agreement between the 27 day running averages when the baseline correction is applied to the model . It is important to note that our point is that the model without the baseline correction is a far more accurate representation of the effects of magnetospheric currents that affect the magnetic field at dst observatories than the kyoto dst index . Figure 4 (bottom) compares the 27 day moving averages once the baseline correction is applied to the model . Now the agreement between the model and the kyoto dst index is very good (correlation coefficient = 0.981). (except for the baseline coefficients, the coefficients of the model were optimized using only data from the years 19952002 .) In addition, we have looked at all the year end final kyoto dst data between 1957 and 2009 and found that the average change at the end of the year (5.9 nt) is more than twice as large as in each of the 2 h before and after (2.5 nt) the end of the year, implying an average discontinuity in the kyoto dst index of between 5 and 6 nt at the end of each year . The largest discontinuity appears to have occurred at the end of 1982 when the kyoto dst index decreased by 20 nt for no apparent reason . For any individual year it is not possible to determine the discontinuity by the change at year's end since some of the change can be physical . But by comparing the model with the dst index as in figure 3, it can be estimated well . We conclude that the process of removing the secular variation from the kyoto dst index inadvertently removes some of the solar cycle variation . Thus, the average value of the kyoto dst index underestimates the modulation effects of the solar cycle on magnetospheric activity and in particular on the ring current . Conversely, if one uses the kyoto dst index to remove the magnetospheric current contribution to the magnetic field to determine the secular variation of the magnetic field, one will also get an incorrect result for the secular variation . It is our claim that the baseline as determined by the standard procedure used to remove the secular variation from the kyoto dst index results in significant offsets (errors) so that the kyoto dst index and especially its longerterm average do not accurately represent the magnetic effects near the equator of the earth of currents flowing above the ionosphere and that the offsets determined by our model are more accurate . There are discontinuities in the kyoto dst index at the end of each year, which are typically larger during solar maximum and which, it is important to note, affect the level of the kyoto dst index for the rest of the year with respect to the previous year . In addition to the discontinuity at the end of each year, a different quadratic function determines the kyoto dst index baseline during that year . The second part of our claim, that our model provides a better baseline offset, is maybe less clear . It is based on the fact that the model (without its own offset term) has no discontinuities at year's end and generally accurately reproduces the kyoto dst index based on the solar wind and now also on the f 10.7 index . It is however possible that if there are additional differences, not captured by the model, in the response of the magnetosphere between solar minimum and solar maximum, that some of the baseline correction determined by the model are corrections to the model's own incorrect response to the solar wind and the f 10.7 index . However, we can think of no reason this should be a large effect . Laying aside the above concern we can say that the average value of the kyoto dst index should be approximately 13 nt more negative for the active year 2003 compared to quiet years 2006 and 2009 (since the values in figure 1 should be subtracted from the dst index to make it agree with model) and by implication, although we have not modeled other solar cycles, it is likely that the kyoto dst index more generally underestimates the solar cycle modulation of the surface magnetic field due to currents flowing above the ionosphere . The model also suggests that in addition to underestimating the solar cycle variation, the solar minimumquiet baseline of the kyoto dst index should be around 15 nt as is clear from figure 1 while the baseline during solar maximum can vary between about 20 nt and more than 30 nt . That is to say during solar minimum when the kyoto dst index reads 0 nt, currents flowing above the ionosphere produce a negative depression of the surface equatorial magnetic field of about 15 nt while during solar maximum when the kyoto dst index reads 0 nt such currents produce a negative depression of the surface equatorial magnetic field between 20 and 30 nt . We care more about the relative change in the baseline offset between solar minimum and solar maximum than we do about its average value . The relative change is much less sensitive to changes in the model parameters than the average value . Nevertheless, we note that our baseline offset has an average value of 21.2 nt (23.0 nt if only the first 11 years are used to avoid two solar minima). This is very similar to the 20 nt constant in the burton equation [burton et al ., 1975], which is based on a much simpler model of the response of dst to the solar wind . Jorgensen et al ., using crres satellite magnetometer data, also conclude that there is an offset in the dst index so that dst = 0 actually corresponds to a ring current that would create a 20 nt depression . Thus, our average baseline offset corresponds well to the effect of the quiet time ring current as determined by others . Our results have significant implications for statistical studies of geomagnetic storms . In such studies, thresholds are often set; such that, for instance, a dst value of less than 30 nt is considered an indication of a geomagnetic storm [e.g., gonzalez et al ., 1999] and that a period when dst remains above 30 nt is regarded as nonstorm [e.g., schiller et al ., 2014]. Since dst can differ by over 10 nt from nonphysical baseline corrections, threshold values should be carefully considered . We get a correlation coefficient of 0.965 between our model and the kyoto dst index for the years 19952002 using only the solar wind and the f 10.7 index (a good but not perfect [e.g., chen et al ., 2011] proxy for the solar euv flux and thus for the ionization of the ionosphere) as input to our model . Such a good correlation would not be possible if the kyoto dst index had large random components . Still, no index is perfect, and others have also tried to remove its defects, both real and imaginary . The index is created in the same manner as the kyoto dst but extends back to 1932 and corrects a few technical issues with the kyoto dst index . Since the secular variation in the dxt index is removed in the same manner as in the kyoto dst index, the problem that we have identified here remains in the dxt index . Karinen and mursula then use annual averages of the dxt index to study the longterm and solar cycle variations of the dxt index . Given that the correction for the secular variation can introduce both random and systematic variations in the annual average of dst and dxt, the results of such studies should be accepted with care . Mursula and karinen and karinen and mursula have also created another index that they call dcx, which they say removes the excessive semiannual variation in the dst index related to the semiannual, nonstorm time behavior of the dst index . We have not noticed an excessive semiannual variation in kyoto dst index and suggest that in the same manner that quiet days during solar maximum are on average less quiet than quiet days during solar minimum, quiet days around the equinoxes are less quiet than quiet days around the solstices . This index removes much of residual sq current system variation that remains in the kyoto dst index . The residual sq current system variation is indeed a problem with the kyoto dst index and can dominate the error in our model during quiet times [temerin and li, 2006]. (much of the highfrequency error seen in figure 3 is probably due to this .) However, dst also removes other regular variations including the excessive semiannual variation and even perhaps some of the solar cycle variation . Also, the magnetosphere has a real diurnal variation since the reconnection rate can depend on the angle between the dipole axis and the solar wind velocity that can also be removed when effects of the sq current system are removed . Svalgaard has suggested using another index (dsv) that depends only on nighttime observations to avoid problems with the sq current system that mostly affects daytime observations . The creation of both the dcx and dst indices seems motivated by a desire to remove the nonstorm component of the dst index . We think that making a hard distinction between storm and nonstorm or quiet times in the dst index is not useful . The dst index and the magnetospheric currents that it reflects are constantly changing in response to the changing solar wind . Level in the 3.5 months of data shown in figure 2, and the number of magnetic storms during this interval depends on ones arbitrary threshold for determining storms: there are number of excursions of the dst index below 50 nt and even more below 30 nt . We have shown that the average value of the kyoto dst index should be approximately 13 nt more negative for the active year 2003 compared to quiet years 2006 and 2009 and that discontinuities in the kyoto dst index at the end of each year have an average value of about 5 nt but may be as large as 20 nt . This implies that the kyoto dst index underestimates the solar cycle variation of currents above the ionosphere that affect the surface equatorial magnetic field . Our study also implies that quiet time currents above the ionosphere produce a negative depression of the surface equatorial magnetic field of at least 15 nt even during solar minimum and as much as 30 nt during solar maximum.
The australian sheep blowfly (lucilia cuprina) is an important ecto - parasite that causes fly strike, which has significant health and welfare, as well as economic, impacts on the sheep industry in australia (sandeman et al ., 2014). The female blowfly is attracted to the sheep by odours, particularly those associated with bacterial infections in damp fleece, and lays eggs (tellam and bowles, 1997). The developing larvae feed on the sheep, causing severe tissue damage, toxaemia, and in some cases, death . The consequent loss of livestock, costs of preventative and curative chemical treatments, and animal welfare issues place significant economic burdens on livestock enterprises (lane et al ., 2015). The blowfly has developed resistance to various classes of chemical insecticides used for its control, including organochlorines, organophosphates, the benzoyl - phenyl urea diflubenzuron (levot, 1995, sandeman et al ., only two preventative blowfly control chemicals, the macrocyclic lactone ivermectin and the cyanopyrimidine dicyclanil, remain effective with no resistance yet reported . There is therefore a need to identify new chemical classes of insecticides, preferably with different target proteins, to control this important parasitic insect . Histone deacetylase inhibitors (hdaci) have been recognised as therapeutic targets in cancer for many years (cairns, 2001), with a number in clinical use or clinical trials as anti - cancer drugs . They have also been studied extensively over recent years for their potential in chemotherapy for parasitic diseases of humans, including malaria, toxoplasmosis, trypanosomiasis, schistosomiasis and leishamaniasis (andrews et al ., 2012a, andrews et al ., 2012b; marek et al ., hdac enzymes have been studied extensively in the model dipteran insect drosophila with respect to their roles in longevity and memory formation (fitzsimons et al ., 2013, 2016), with a drosophila model providing experimental evidence to highlight hdaci as potential therapeutics for the treatment of huntington's disease (sharma and taliyan, 2015). However, only a single study has reported the insecticidal activity of an hdaci against this fly species, with pile et al . (2015) who showed that trichostatin and suberoylanilide hydroxamic acid (saha) were able to inhibit the development of sheep blowfly larvae in vitro . That report also highlighted similarities and differences in amino acid sequences of blowfly and human hdac enzymes, with differences particularly noted between species for the class ii enzymes hdac4 and 6, and the class iv hdac11, raising the possibility of identifying insect - specific inhibitors . The present study expands on our earlier report of insecticidal activity for trichostatin and saha (kotze et al ., 2015) by examining other hdaci with different chemical structures and mechanisms of action . We focus on hydroxamic acids since these are the best known group of hdaci, but also include inhibitors with different chemical components, such as benzamides, thioesters, thiolates, disufides, cyclic depsi- and tetra - peptides, fatty acids, and sesquiterpene lactones (table 1). We measure the effects of these hdaci on the development of blowfly larvae (larval growth rate and pupation rate) and on the hdac enzyme activity of nuclear protein extracts prepared from blowfly eggs . We also compare these results with reported inhibitory activities against human hdac enzymes as an initial step towards identification of insect - specific inhibitors . The l. cuprina used in this study were from the laboratory reference drug - susceptible ls strain, derived from collections made in the australian capital territory (canberra, australia) over 40 years ago . This strain has been maintained in a laboratory since that time (in canberra for 30 years, and then at csiro and university of queensland laboratories in brisbane for the last 10 years), and has no history of exposure to insecticides . Adult flies were maintained at 28 c and 80% relative humidity with a daily photoperiod of light 16 h and dark 8 h. adults were fed a diet of sugar and water, while larvae were raised on a wheatgerm culture medium (tachibana and numata, 2001). Protein meals (bovine liver) were provided on days 4 and 8 after adult eclosion in order to prime adult flies for subsequent egg - laying . For provision of eggs for bioassays, liver was placed into cages of gravid flies for a period of two hours (12 p.m. until 2 p.m.). The liver was then removed and kept at room temperature overnight . At 10 a.m. the next morning hdaci were synthesized by reported procedures or obtained from commercial sources (table 1). Stock solutions for use in larval bioassays were prepared in ethanol at a concentration of 1 mg / ml . In cases where the compound did not dissolve at this concentration the solutions were further diluted 2-fold with ethanol until no precipitate was evident (to give stocks at 0.5 or 0.25 mg / ml). Exceptions were cudc-907 and mc1568 which required dilution to a concentration of 0.05 mg / ml . The commercial insecticide stocks used as controls were prepared at 1 mg / ml in water (cyromazine and dicyclanil) or acetone (diflubenzuron). Stock solutions of hdaci for use in nuclear extract hdac enzyme assays were prepared at 1 mg / ml in dmso . The effects of hdaci on the growth of blowfly larvae was assessed using a bioassay system in which larvae were allowed to develop on cotton wool impregnated with the compounds at various concentrations (modified slightly from kotze et al ., 2014). Briefly, 4 ml aliquots of hdaci or commercial insecticide solutions were added to cotton wool plugs and the solvent (4 ml of either ethanol, acetone, or water) was allowed to evaporate overnight . Control containers were prepared by addition of 4 ml of the relevant solvent to the cotton wool . The next day (day 0 of the assay), a sheep serum - based medium (80 g / l yeast extract (merck), 1.6 mg / ml tylosin (sigma) in lamb serum (life technologies) buffered with 35 mm kh2po4, ph7.5) was added to the cotton wool, and groups of 50 freshly - hatched larvae (prepared as described in section 2.1, above) were placed onto the cotton wool . The assay pots were placed at 28 c . In order to calculate mean larval weight at the beginning of the drug exposure period, two groups of 100 larvae were collected, blotted dry on paper towel, weighed and discarded on day 0 . After 24 h (day 1), 3 larvae were removed from each container, weighed, and discarded . The remaining larvae were fed with 1 ml of nutrient medium on day 1, and then 2 ml on each of days 2 and 3 . Late on day 4, the containers were placed into larger pots with a layer of sand at the base to serve as a medium for pupation, and returned to the incubator . Each experiment consisted of a single container at each concentration of hdac inhibitor or insecticide, alongside 4 control assays . The effect of the compounds on larval development was defined in two ways: i)larval weight gain in first 24 h; the total weight gain of the 3 larvae sampled on day 1 was expressed as a percentage of the mean of the weight gain of the 3 larvae sampled from each of the 4 control containers (weight gain was calculated by difference using weight on day 1 and the mean weight of larvae on day 0);ii)pupation rate; the number of pupae in each drug - treated container was expressed as a percentage of the mean number of pupae in the 4 control containers . Larval weight gain in first 24 h; the total weight gain of the 3 larvae sampled on day 1 was expressed as a percentage of the mean of the weight gain of the 3 larvae sampled from each of the 4 control containers (weight gain was calculated by difference using weight on day 1 and the mean weight of larvae on day 0); pupation rate; the number of pupae in each drug - treated container was expressed as a percentage of the mean number of pupae in the 4 control containers . The larval weight and pupation rate dose - response data were analysed with graphpad prism software using non - linear regression, with the variable slope option selected, in order to calculate ic50 values (with 95% confidence intervals) representing the concentration of inhibitor required to reduce the larval weight gain or pupation rate to 50% of that measured in control (no drug) treatments . Nuclear extracts were prepared from blowfly eggs (0.5 g) using a nuclear extraction kit (millipore, usa) following the manufacturer's protocol with some modifications . The chorion was removed by soaking for 80 s in a solution of bleach (2% v / v), followed by centrifugation to sediment the eggs . Complete mini protease inhibitor (roche, basel switzerland) in pbs was added to the washed eggs before disrupting them by hand with a plastic pestle . The disrupted eggs were centrifuged at 250 g for 1 min at 4 c, and supernatant removed . The egg cell pellet was washed with 1000 l of ice cold pbs, resuspended by inversion, centrifuged at 1000 g for 5 min at 4 c, and the supernatant removed . The cells were then disrupted by drawing 5 times through a 21 g needle fitted to a 1 ml syringe . The suspension was centrifuged at 8000 g for 20 min at 4 c, the supernatant removed and discarded, and the pellet retained (nuclear portion). The nuclear pellet was resuspended in 2/3 of the original cell pellet volume of ice cold nuclear extraction buffer (containing 0.5 mm dtt and protease inhibitor cocktail, millipore, temecula). The solution was placed on low speed roller for 1 h at 4 c, then centrifuged at 16000 g for 5 min at 4 c, and the supernatant (the nuclear extract) transferred to a new tube . The protein concentration was measured by the method of bradford (1976) using the bio - rad protein assay reagent, and bovine serum albumin as a standard . The extract was then aliquoted into separate tubes, snap - frozen in liquid nitrogen, and stored at 80 c . A fluorometric assay kit (sigma - aldrich, usa) was used to measure hdac enzyme activity in blowfly nuclear extracts, as described in the kit instructions, except that the volumes of all reagents were reduced to give a total assay volume of 27.5 l . Control assays were also run in the presence of 1.25 m trichostatin in order to calculate the amount of fluorescent product that was derived from a trichostatin - inhibitable reaction, that is, the amount of product derived from the action of hdac enzymes alone . The assay was performed using a series of at least 4 serially - diluted working solutions of each hdaci . The fold dilutions used to generate each working solution series varied from 2fold to 10-fold, and were set (based on initial dose - finding experiments) in order to provide a dose response curve consisting of 46 data points . The% inhibition of hdac activity was calculated for each concentration of hdaci added to the reaction . The enzyme assay dose - response data were analysed with graphpad prism software using non - linear regression, with the variable slope option selected, in order to calculate ic50 values (with 95% confidence intervals) representing the concentration of inhibitor required to reduce the hdac activity of the nuclear extract by 50% . We performed a non - parametric (spearman) correlation analysis in graphpad prism in order to examine the relationship between the effects of hdaci in inhibiting blowfly larval development and inhibiting nuclear extract hdac enzyme activity . In addition, in order to examine the relationship between the blowfly bioassay data and the reported inhibitory effects of the hdaci against specific human hdac enzymes, we performed a correlation analysis using the bioassay data and ic50 values reported in the scientific literature for the hdaci against human hdac enzymes (see supplementary table 1). While blowflies are known to possess hdac1, 3, 4, 6 and 11, (kotze et al ., 2015), the analysis was only performed with human hdac1, 3, 4 and 6 as insufficient inhibition data was available for an analysis of inhibitory effects on human hdac11 . For the correlation analysis, we grouped hdac 1 and 3 together as class i hdac enzymes, and hdac4 and 6 together as class ii hdac enzymes . Forty hdaci compounds were investigated for inhibition of the growth of blowfly larvae, with their activities reported in table 2 as inhibition of larval weight gain and pupation (g / assay). For comparison, the most potent inhibitor of blowfly larval growth was the depsipeptide romidepsin, which was more potent, or as potent as, the commercial insecticides: 10-fold more potent than cyromazine, 2-fold more potent than diflubenzuron, and equipotent with dicyclanil (table 2, fig . 1, fig . 2). The most potent hydroxamic acids were trichostatin, cudc-907, al179 - 3b and ar-42: approximately 10-fold less potent than cyromazine, and approximately 50100fold less potent than diflubenzuron and dicyclanil . Also showing marked activity (ic50 <100 g / assay) were the thioester compound kd5170, the disulfide compound psammaplin a (which is a prodrug that forms a thiolate much like romidepsin), and the cyclic tetrapeptide apicidin . Many of the compounds, including 13 of the hydroxamic acids, the two fatty acids (valproic acid and an-9), and the single sesquiterpene lactone (parthenolide) showed little or no insecticidal activity (ic50> 1000 g / assay). Comparisons between the larval weight gain and pupation ic50 for the commercial insecticides showed that the two values were within 2-fold of each other . For 7 of the 8 most active hdaci (larval ic50 <100 g / assay, fig . 2), the variation between the larval and pupation ic50 values was also within a 2-fold range . The two values were approximately equal for cudc-907 and ar-42, while within 2-fold for trichostatin, al1179 - 3b, romidepsin and kd5170 . On the other hand, the hdaci were also investigated for inhibition of hdac activity in nuclear extracts from blowfly eggs (table 3), with representative dose - response curves shown in fig . 3 (some of the compounds shown in table 1, table 2 were not examined in nuclear extract assays as insufficient material was available). As with the insecticidal assays, romidepsin was the most potent inhibitor of hdac activity . This compound was approximately 600-fold more potent than the second most - active compound, quisinostat, and about 1000-fold more potent than trichostatin . The hydroxamic acids that were the most active in the blowfly larval bioassay were among the most potent enzyme inhibitors (ic50 0.0160.212 m for trichostatin, cudc-907 and ar-42). A number of hydroxamic acids that were significant hdac enzyme inhibitors in the nuclear extracts (ic50 <0.3 m) had low potency in the larval bioassay (e.g. Panobinostat, givinostat, belinostat: larval ic50 295, 477, and 740 g / assay, respectively). Among the other compounds highlighted above for their insecticidal activity (from fig . 2), all showed significant potency in inhibiting the hdac enzyme activity of the nuclear extract (all ic50 <1 m). The relationship between larval bioassay ic50 and nuclear extract hdac inhibition ic50 is shown in fig . 4 (fig . 4b shows data points with extract hdac inhibition ic50 <2.0 m only). 4a), revealed that the two assay parameters were significantly correlated (spearman correlation coefficients shown on figure panels). Despite this, some differences between the two measurements were apparent, with larval weight ic50 values of 1000 (n = 14) corresponding to a range of nuclear extract activities from 0.032 m (cudc-101) to> 100 m (six compounds). Importantly, low larval weight ic50 values (<100 g / assay) did not occur alongside high nuclear extract ic50 . 4b illustrates this, with the most active insecticidal compounds all being potent inhibitors of hdac activity in blowfly nuclear extracts (ic50 <0.5 m). We also examined the relationship between published ic50 values for inhibition of human hdac enzymes by the hdaci used in this study with their activity in inhibiting blowfly larval development . The analysis was restricted to just the human hdacs that corresponded to the class i and class ii hdac enzymes present in the blowfly, namely hdac1 and 3 (class i) and hdac4 and 6 (class ii). The published data on the inhibition of human hdac11 (corresponding to the other hdac present in the blowfly) was not extensive enough with respect to the hdaci examined in the present study (see supplementary table 1) to allow for a separate analysis of this class iv hdac . The relationship between the blowfly bioassay data for each hdaci and the reported enzyme inhibition ic50 values against the class i and ii human hdac enzymes are shown in fig . The two parameters were significantly correlated for the class i enzymes, but not for the class ii enzymes . However, even though a significant correlation existed for class i enzymes across the whole data set, a number of compounds that were potent inhibitors of the human class i enzymes showed no insecticidal activity (ic50> 1000 g / assay). The present study has examined the ability of a number of known hdaci to inhibit the growth and development of blowfly larvae, and correlated this effect with their ability to inhibit the hdac activity of nuclear extracts prepared from blowfly eggs . There was a significant correlation, suggesting that their insecticidal activity was likely due to the inhibition of blowfly hdac enzymes . Romidepsin was a very potent inhibitor of both blowfly larval growth and blowfly hdac activity, the potency being equivalent to or greater than commercial blowfly insecticides . In addition, we have shown that a number of other hdaci have significant insecticidal activity against blowfly larvae, including hydroxamic acids (trichostatin, cudc-907, al1179 - 3b, ar-42), a thioester (kd5170), a disulphide (psammaplin a) and a cyclic tetrapeptide with a zinc - binding ketone (apicidin). While these hdaci validate the concept of a potentially valuable new target for insecticides, we are not advocating the use of the particular compounds reported herein as commercial insecticides . They would be too expensive to be economically viable for any livestock or agronomic production setting . Moreover, most of the more potent hdaci described are also potent inhibitors of human hdaci (ic50 nm - m) and might prove cytotoxic in sheep and unacceptable in terms of human consumption of sheepmeat . Hence, while our demonstration of the potent insecticidal activity of a number of hdaci helps to prove the concept that hdaci may be effective insecticides, issues associated with cost of production and target pest selectivity need to be solved next . Romidepsin is a prodrug that is first activated by reduction of its disulfide to the free thiol that can then bind to the catalytic zn in hdac enzymes . . The higher potency of romidepsin involves either a highly complementary fit of the conformationally constrained cyclic depsipeptide component of romidepsin with the enzyme, or higher metabolic stability than the hydroxamates . Apicidin is another compound with significant insecticidal activity (table 2) which also has a rigid cyclic tetrapeptide component that adds affinity to the relatively weak interaction between its ketone component and zinc . (2015) found that romidepsin inhibited the growth of asexual stage plasmodium falciparum (ic50 0.1 m), the bloodstream form trypanosoma brucei parasites (ic50 0.035 m), and was a potent inhibitor of hdac enzyme activity in p. falciparum nuclear extracts (ic50 0.9 nm). In contrast, most hydroxamic acid based inhibitors derive their affinity from zinc chelation which sometimes compensates for a suboptimal fit between the remaining features of the inhibitor and the enzyme active site . The 31 hydroxamic acids examined here have considerable structural diversity and are mostly potent inhibitors of human hdacs . They show quite a range of inhibitory potencies against blowfly larval growth over two log units (table 2). Most of the hydroxamate - based inhibitors were derived from 4-aminopyrimidine or 4-aminobenzene hydroxamic acids, which confer an extended linear shape to the fragment projecting towards zn in the enzyme . Other active inhibitors with a linear structure due to an aromatic group in conjugation with a double bond and hydroxamate are the cinnamic acid hydroxamates, panobinostat & pracinostat . The potent suberoylhydroxamates (al1179 - 3b & pg50) have a more flexible linear zinc - binding extension like the similarly flexible but simpler parent compound saha, but exhibit superior activity attributed to their branched capping group that likely makes multiple interactions with the enzyme . Pg50 was developed as a selective inhibitor of human hdac6 (gupta et al ., 2010), however it seems to be a class i hdaci in the blowfly possibly suggesting its capping groups are too small to influence selectivity as the other hydroxamate inhibitors known to specifically inhibit human hdac6 (tubacin & tubastatin a) were inactive in the blowfly bioassay . The reasons why other hydroxamates were inactive is not clear, but they do show how selectivity between highly homologous enzymes can be achieved, in this case away from blowfly and towards human . In principle clues derived from the capping cyclic peptide groups away from the zinc - binding moieties of romidepsin and apicidin may steer the development of new compounds with greater potency and selectivity for the target enzyme to make better and safer insecticides . Firstly, effective insecticides must kill, or inhibit the growth of early stage larvae before they can damage the host . Secondly, where the initial effects are inhibitory rather than lethal, they must persist over at least several days and then kill the larva to prevent it recovering and developing to damage the host . A comparison of the two bioassay ic50 values is informative with respect to these time course considerations . The commercial insecticides show a pupation ic50 that is similar (within two fold) to the 24 h weight gain ic50, consistent with the larvae not recovering from an initial growth inhibition phase . This was also observed for seven of the eight hdaci highlighted in fig . 2 . Apicidin on the other hand showed a pupation ic50 value almost 6-fold greater than the weight gain ic50, indicating some recovery of larvae after the initial inhibitory effects on growth . A number of compounds showed potent inhibition of the nuclear extract hdac activity, but only low or no activity in the larval bioassay (for example: nuclear enzyme assay cay10603 ic50 0.165 m, cudc-101 0.0317 m vs larval bioassay ic50 this is likely due to poor uptake or low stability of the compounds in the larval assay . There are likely to be differences between the various compounds examined in terms of uptake across the larval cuticle (trans - cuticular uptake) and across the intestinal membranes (following ingestion), as well as access to the cellular target following uptake . Some of the compounds are likely to be metabolised to a greater degree than others by the blowfly xenobiotic - detoxification systems, which include esterases (campbell et al ., 1997), cytochromes p450 (kotze, 1993) and glutathione transferases (kotze and rose, 1987). Potency against human class i hdac enzymes generally correlated with insecticidal activity, but some potent inhibitors of human class i hdac (ic50 <0.10 m) showed no insecticidal activity . This may be due to factors associated with uptake and stability of the compounds in the bioassay, as well as differences in the intrinsic level of interaction of the compounds with the human enzymes compared to the equivalent blowfly hdac enzymes . (2015) described some differences in the amino acid residues between the human and blowfly class i hdacs, with catalytic domain amino acids showing 86% and 73% identities between human and blowfly hdac1 and 3, respectively . The relationship between inhibitory effects of hdaci on human class ii hdacs and their insecticidal activity was poor, with no significant correlation between the two parameters . The catalytic domain amino acids differ to a much greater extent between the human class ii hdacs and their blowfly equivalents compared to the class i comparisons, with% identities of 61%, 47% and 50% for hdac 4 and the two catalytic domains of hdac6, respectively, between the human and blowfly (kotze et al ., 2015). Hence, hdaci of human and blowfly class ii enzymes may show a lower level of relatedness than among inhibitors of class i enzymes from the two species . The lack of correlation for class ii hdacs may be favourable for potential identification of more insect - specific hdaci that interact specifically with the blowfly class ii enzymes, while showing less inhibition of the human class ii enzymes . However, more information on the different roles played by the blowfly class i and ii hdac enzymes is required before a preferred target hdac class or individual enzyme can be determined . (2006) found that rnai mediated silencing of drosophila hdacs 1 and 3 resulted in inhibitory effects on growth curves for drosophila schneider (s2) cell lines, whereas silencing of hdacs 4, 6 and 11 did not inhibit cell growth, suggesting more important roles for the two class i enzymes in cell viability . (2010) reported that drosophila hdac6 loss - of - function mutant flies were viable and fertile, suggesting that this enzyme may not be essential for the development of this fly species . In conclusion, the present study shows that hdaci from various chemical groups can substantially inhibit the development of blowfly larvae . In particular, romidepsin was at least equipotent with the major commercial blowfly insecticides, supporting the concept of inhibiting blowfly hdac enzymes to produce new insecticides for preventing infection by sheep blowfly, and to potentially control other insects . There is a great deal of interest currently in developing hdac inhibitors for use in chemotherapy against other human parasitic disease malaria, toxoplasmosis, trypanosmiasis, schistosomiasis and leishmaniasis (andrews et al ., 2014, kelly et al ., 2012, hansen et al ., 2014, engel et al ., 2015, marek et al ., 2015) a focus of these studies is the identification of hdaci that show selectivity for the parasite hdac enzymes over the human enzymes . Similarly, further work on developing hdac inhibitors as potent insecticides could focus on identifying insect - specific inhibitors, but at the very least should focus on producing hdaci that are cheap to manufacture and market as prospective insecticides.
Chromosomal abnormalities affect about 0.5% of living newborns, and are associated with congenital malformation, cognitive defects, learning disabilities, seizures, etc . Cytogenetic techniques can diagnose chromosomal abnormalities, and investigate the possible etiology of birth defects . It is important to know the clinical data of chromosome abnormalities in order to explore the corresponding relationships between the phenotypes and certain chromosome abnormalities, and increase the evidences of initial clinical indications of these types of disorders in different ages . Furthermore, the cytogenetic outcomes can guide medical professionals the optimal treatment, social function training, and predicting the possible prognosis . Our tertiary care referral center previously reported the results of cytogenetic survey from 1996 to 2010, which allowed us to closely gain insight into the incidence and distribution of the cytogenetic abnormalities in outpatient children suspected with congenital disorders . The purpose of the present study was to collect data among children who were suspected with chromosomal disorders from january 1, 2011 to march 31, 2014 in the children's hospital, zhejiang university, and tried to establish and update our previous database of common chromosomal anomalies that could be useful for genetic counseling and reminding the medical professionals which kind of patients should be transferred to genetic analysis . We collected children who were suspected with chromosomal disorders from january 1, 2011 to march 31, 2014 since this study was an update to the previous report by the same team in the children's hospital, zhejiang university . The informed consents were obtained from children's parents / guardians or other legally authorized representatives before the chromosome analysis preparation, including clinical interview of the medical histories and blood sample collections . The clinical features were recorded and the blood sample were collected, and then the blood samples were sent to the medical biology and genetic department laboratory for cytogenetic analysis at zhejiang dian diagnostics, which is an independent third - party medical diagnostic service institution . According to the reasons for referral for cytogenetic analysis, we divided them into 4 groups: group 1, who presented with specific clinical stigmata (such as up slanting palpebral fissure, prominent epicantic folds, micrognathia, etc . ); group 2, who had speech or motor developmental delay, or both, or learning disabilities; group 3, who presented with congenital genitourinary defects (including ambiguous genitalia, abnormality of male external genitalia, concealed penis, cryptorchidism, shield chest, widely spaced nipples and amenorrhoea, etc . ); and group 4 (miscellaneous group, including obesity, congenital heart diseases, primary seizures and other indications not listed in the above three groups). For those who presented with both specific clinical stigmata and genitourinary defects we would put them into 1 group according to the main complains of their main problems . For routine cytogenetic analysis, 0.5 to 1.0 ml peripheral blood samples were collected from the patients and stored into heparinized test tubes . The karyotypes were determined by g - banding using trypsin and giemsa (gtg). At least 30 cells were routinely analyzed; in cases of mosaicism, this number was increased to approximately 100 metaphases . The karyotypic descriptions were reported according to the international system for human cytogenetic nomenclature recommendations (iscn, 1995). The percentage of abnormal cases in each group and the distribution of the numerical and structural abnormalities were determined . We used the chi - squared test to evaluate the detection rates and types of chromosomal anomalies among groups according to different classification criteria . We collected children who were suspected with chromosomal disorders from january 1, 2011 to march 31, 2014 since this study was an update to the previous report by the same team in the children's hospital, zhejiang university . The informed consents were obtained from children's parents / guardians or other legally authorized representatives before the chromosome analysis preparation, including clinical interview of the medical histories and blood sample collections . The clinical features were recorded and the blood sample were collected, and then the blood samples were sent to the medical biology and genetic department laboratory for cytogenetic analysis at zhejiang dian diagnostics, which is an independent third - party medical diagnostic service institution . According to the reasons for referral for cytogenetic analysis, we divided them into 4 groups: group 1, who presented with specific clinical stigmata (such as up slanting palpebral fissure, prominent epicantic folds, micrognathia, etc . ); group 2, who had speech or motor developmental delay, or both, or learning disabilities; group 3, who presented with congenital genitourinary defects (including ambiguous genitalia, abnormality of male external genitalia, concealed penis, cryptorchidism, shield chest, widely spaced nipples and amenorrhoea, etc . ); and group 4 (miscellaneous group, including obesity, congenital heart diseases, primary seizures and other indications not listed in the above three groups). For those who presented with both specific clinical stigmata and genitourinary defects we would put them into 1 group according to the main complains of their main problems . For routine cytogenetic analysis, 0.5 to 1.0 ml peripheral blood samples were collected from the patients and stored into heparinized test tubes . The karyotypes were determined by g - banding using trypsin and giemsa (gtg). At least 30 cells were routinely analyzed; in cases of mosaicism, this number was increased to approximately 100 metaphases . The karyotypic descriptions were reported according to the international system for human cytogenetic nomenclature recommendations (iscn, 1995). The percentage of abnormal cases in each group and the distribution of the numerical and structural abnormalities were determined . We used the chi - squared test to evaluate the detection rates and types of chromosomal anomalies among groups according to different classification criteria . There were totally 4129 children referred to cytogenetic analysis from january 1, 2011 to march 31, 2014, including 1857 boys and 2272 girls . The average age was 51.7 months, median age was 33 months, and age ranged from 1 day to 18 years and 11 months old . The ratios between cases referred for cytogenetic analyses and total outpatient visits were 1:1607 (1036/1,665,048) in 2011, 1:1364 (1328/1,812,521) in 2012, 1:1318 (1448/1,908,152) in 2013, and 1:1329 (317/421,532) in first quarter of 2014, respectively . There was no statistical difference between the referral ratios in these years by chi - squared test (= 0.03, p = 0.99). But compared to previous report, the referral ratios were higher than that in 2010 (= 448, p <0.001). There were 769 children who had chromosome abnormalities, accounting for 18.62% of all referral cases . The detection rates of abnormalities were 19.66% (365/1857) for boys and 17.78% (404/2272) for girls the detection rate of autosomal anomalies was higher in boys (16.6% vs 12.2%, = 16.6, p <0.001), but the detection rate of sex - linked chromosomal anomalies was higher in girls than that in boys (5.6% vs 3.0%, = 17.2, p <0.001). Rate of chromosome abnormalities in boys and girls in different age groups according to referral causes, there were 4 groups set . The percentages of chromosome abnormalities for groups 1, 2, 3, and 4 were 59.1%, 10.6%, 7.9%, and 13.1%, respectively . In group 1, in which children presented with specific clinical stigmata, the detection rate of chromosome abnormalities was highest . While for group 3, in which children presented with congenital genitourinary defects, the detection rate of chromosome abnormalities was lowest (fig . Totally, 359 cases were detected with down syndrome, 76.9% (276 cases) of which were found before their age of 1 year old, and 90.5% (325 cases) were found before their ages of 3 years old . There were 81 children with turner syndrome (ts), 76.5% of which (62/81) were diagnosed after 6 years old . Almost half of the children with ts had the karyotypes of mosaicism (table 2). Types of chromosomal abnormalities according to age when children were transferred to do cytogenetic analysis, the detection rates were higher in neonates and infants than that in others (fig . Further analysis showed that most of chromosome abnormalities were trisomy 21, accounting for almost half of all the abnormalities (fig ., we presented the cytogenetic results of 769 cases in detail (table 2). Four hundred two (19.5%) were confirmed by cytogenetic analysis and their results were consistent with primary diagnosis . In the other 2070 children with unknown causes, 326 (15.7%) this investigation showed the total detection rate of chromosome abnormalities was 18.62% in the cases suspected with congenital disorders, which was lower than that in the previous report undertaken by the same study group from 1996 to 2010 . In recent 2 decades, it could only indicate the occurrence rate of 1 disease in the group of suspected with 1 type of disease in 1 center . The data from these patients were compared with that from our previous published group of children (19962010) from the same children's hospital . We found that the ratios between cases referred for cytogenetic analyses and total outpatient visits were increased from 2011 to 2014 . Compared to the previous report, these present ratios were higher than that before 2006, and equal to that post-2007 . This result indicated that the professionals had more awareness of transferring the children with clinical manifestations of unknown causes to do cytogenetic analysis . The most notable feature of these types of disorders is diagnostic difficulty since they present diverse clinical pictures . Physical examination, radiographic studies, and histologic investigation may prove to be equivocal . The data from table 1 showed that the detection rate for the children <1 year old was higher than that for other older age groups ., we should take it into consideration that the percentage of trisomy 21 was 76.9% in total chromosome abnormalities in 0 to 1 year group, and 90.5% in 0 to 3 years group . These data indicated that professionals had the experiences to identify this common syndrome, even when patients were very young . However, for the most common sex - linked chromosome abnormality of ts, only 5 children were diagnosed in 1 year old, accounting for 6.2% of total diagnosed cases (more than 2/3 of them (31/81) were identified until children had more symptoms in their adolescent stages . This indicates the clinical doctors were not familiar with ts, especially for those who were very young . If we found the patients in their early stages, we could treat them timely, which would greatly improve their prognosis, including final stature and sexual development . Therefore, it was significant to do more training with the diverse features of these type of chromosome disorders, which would contribute to finding them early, and being treated timely . If we compared the detection rate according to the reasons for referral for cytogenetic analysis, we found that the group 1 which presented with specific clinical stigmata had highest detection rate of 59.1% . Among them, 91.9% (330/359) of down syndromes, 25.0% of tss were found . We speculated that, as mentioned above, our professionals were familiar with down syndrome, but were not with ts . We should take the factor of detection resolution into consideration that might lead to low detection rate . Normal development of the genitourinary (gu) tract is a complex process that frequently goes abnormal . In male children, the most frequent congenital gu anomalies are cryptorchidism (14%), hypospadias (1%), and micropenis (0.35%). Current evidences suggested that monogenetic changes contributed to the congenital genitourinary defects, which could be detected at dna levels or fluorescence in situ hybridization (fish) or array comparative genomic hybridization (acgh) rather than chromosomal analysis . The professionals could transfer these children to do next - generation sequencing or microarray and promote the detection rate . Most of them were transferred to do cytogenetic analysis because they were found with abnormal appearance of the external genitalia, or without second sexual features in their ages of more than 12 years . Thus, the percentages of sex - linked chromosome abnormalities in children older than 6 years were higher than those younger than 6 years (10.9% vs 52.7%). Presently, most professional organizations have recommended more higher resolution tests for children suspected with chromosome abnormalities, as acgh testing . But the acgh testing is not popularized among general hospitals in china, and which is more expensive . Secondly, we did not establish an interaction between a specific type of chromosome abnormality and clinical features . Actually, because of the miscellaneous information, the clear relationship is not established . As an alternative designation, we divided these clinical features into 4 groups to define an extensive association of clinical features with chromosome abnormalities . This study demonstrated the detection rates of chromosome abnormalities in children who were suspected with chromosomal disorders . There were 769 children who had chromosome abnormalities, accounting for 18.62% of all referral cases . Among the affected, the percentage of sex - linked chromosomal abnormalities in all was 23.8% . Combined with previous report, we established a database of common chromosomal anomalies and the clinical features and remind the medical professionals what kind of patients should be transferred to genetic analysis.
Although some recent studies have shown declines in the prevalence of hearing impairment in recent generations,1 2 according to world health organization global estimates, it still represents the most common sensory disorder in the world, with 328 million adults having disabling hearing loss (hearing thresholds above 40 db), which can impair their quality of life and potentially result in feelings of isolation, loneliness, and frustration due to communication difficulties, economic participation, and lack of access to services.3 4 however, even in developed countries, it is estimated that less than 1 in 40 people who need hearing aids have access to them.3 in 2004, with the establishment of the national hearing health care policy, the brazilian government, through the unified health system (sus), via the organization of state hearing health networks, made it possible for the population to have access to the service levels of basic, medium, and high - complexity audiological care.5 in parallel, the processes of globalization and urbanization have resulted in changes in lifestyle . Inactivity and unhealthy diet have resulted in an epidemic of excess weight,6 associated with stress, smoking, and alcohol abuse . In turn, these conditions have led to an increased prevalence of chronic diseases, the most common being cardiovascular, which includes hypertension and hypercholesterolemia; osteoarticular; psychiatric disorders; chronic respiratory diseases; diabetes; kidney disease; and cancer.6 7 8 9 all are significantly more prevalent in women.10 certain chronic degenerative diseases may contribute to the onset and worsening of hearing loss via different pathophysiological mechanisms.7 8 according to the national sampling policy of households (pnad), conducted by the brazilian institute of geography and statistics in 2008, hypertension is the most prevalent chronic disease in both sexes.10 11 12 it evolves from increased blood viscosity and microcirculatory failure that can compromise the auditory and vestibular systems at the peripheral and central levels . This is aggravated by hypercholesterolemia, which affects one - third of individuals over 45 years of age.6 7 13 diabetes, although the seventh most prevalent chronic disease in the same pnad statistics, showed the strongest growth, up 37%, compared with the prevalence described in the 2003 study.6 9 although there is still no consensus on the pathophysiology of hearing damage caused by diabetes, it is suggested that changes in the metabolism of lipids and glucides could be related to the development and/or aggravation of not only hearing loss but also tinnitus and balance disorders because of reduced blood flow and disability in the transport of nutrients, due to diabetic microangiopathy and secondary degenerative neuropathy affecting peripheral and central auditory pathways.7 14 15 such situations, besides contributing to the concomitant hypoacusis with other otologic complaints,16 17 18 increase the progression of hearing loss and are of the utmost importance when selecting and fitting hearing aids . Several studies have shown the high concurrency of otologic symptoms in hearing aid users, especially tinnitus, dizziness, itching, and ear pain . Although results vary, tinnitus symptoms have been reported by at least 70% of the subjects in most polls, followed by itching and/or dizziness in 50% of individuals.19 20 21 tenrio et al found that 100% of a sample for hearing aid wearers had at least one associated symptom.12 if on the one hand adaptation of the hearing aids has improved some of these symptoms,20 21 22 23 then on the other hand, hearing aid user may be related to a worsening clinical picture in cases of allergic contact dermatitis24 25 and even worse tinnitus if the device has no ventilation or is an open mold.22 such symptoms can compromise the benefits of hearing aid use . Recently, andersson et al found that hearing aid users with tinnitus had worse responses than the group without tinnitus in unfavorable listening situations (low signal - to - noise ratio), generating more complaints than improvements with the device.26 the impact of hearing loss in the world, the prevalence of systemic diseases and otologic symptoms in the adult population, in addition to the increase in number of sus users getting hearing aids show the importance of developing protocols for integrated assessment of candidates for fitting . Greater attention should be paid to device selection and adaptation to better characterize and manage hearing loss and user expectations and otolaryngologic symptoms associated with systemic comorbidities to optimize the hearing aid fitting process . This study aims to compare the occurrence of otologic complaints, systemic diseases, and effective use of hearing aids in men and women with deafness . This is an exploratory, descriptive, cross - sectional study, approved by the research ethics committee under the number cep/027/2008 . All participants signed a consent form authorizing the use of the collected data . Between march and september 2013, during the development of this research, 278 patients using hearing aids were followed by the otolaryngology service at a hearing health service center accredited by sus in curitiba, brazil . For this study, individuals were selected who fit the following criteria: over 50 years old with a diagnosis of hearing loss, hearing aid use for at least the past 6 months, having complaints about the adaptation and/or functioning of the device . Following this criteria the sample all subjects were evaluated by an ear, nose, and throat (ent) physician and audiologist . Gender, age, otologic complaints, presence of associated systemic diseases, and otoscopy findings were variables taken into account for the ent assessment . In the clinical assessment, the type, degree, and configuration of hearing loss observed in pure tone audiometry were considered, as well as the type of hearing aid worn . For the audiometry, an itera audiometer (madsen, denmark), calibrated in a soundproof booth according to the standards required by the federal council of speech - language pathology, the subjects were divided into two groups for purposes of comparison and analysis of results: group a, made up of women, and group b, by men . The results were statistically analyzed using the chi - square test (to compare possible differences between observed and expected frequencies for an event) and the difference in proportions test (to compare differences between two populations), with 0.05 as the significance level . In the period that data were collected, 278 people presented for a follow - up visit at an accredited clinic . Of these, 61 (21%) subjects reported otologic or operational problems with their devices . The sample was composed of 61 individuals, 35 (57%) women and 26 (43%) men . The age in group a (women) ranged from 53 to 85, with a mean of 72, and in group b (male), from 53 to 82, with a mean of 69 . A family history of hearing loss was reported in 41% of subjects, 13 women and 12 men . There was a predominance of widows in group a (51%), followed by married (37%) and single (11%), and group b was predominantly married (81%), followed by divorced (11%) and widowers (8%). Both genders were mostly made up of retirees or pensioners (71% of the women and 85% of the men). When talking about hearing aids, three individuals in group a used unilateral prosthesis, four used intracanal hearing aids, and 53 (86%) used behind - the - ear (bte) prosthesis with silicone molds . In the group b, two individuals used prosthesis in one ear, six used intracanal hearing aids, and 48 (78%) used bte prosthesis with silicone molds . The chi - square test predominantly showed mild to moderate hearing loss with descending slopes . About 80% of subjects had comorbidities, and the data are shown in table 2 . Note: using chi - square test, there is significant difference in the degree of loss (p = 0.0103) and configuration (p = 0.0422). Abbreviation: n / a, not applicable . Note: the number of citations is greater than the number of respondents because some individuals had more than one associated morbidity . Using the difference of proportions test, there is significant difference between the number of morbidities for hypercholesterolemia and hypertension . The majority of the sample (92%) had bilateral fitting of hearing aids, with the same percentage represented in both groups . The hearing aid use profile is shown in table 3 . Considering only the totals of continuous and discontinuous use, the chi - square test at a significance level of 0.05 showed no significant dependence between usage type between the two genders . Fourteen subjects (22%) reported little use of hearing aids, either because of otologic problems associated with deafness or malfunction of the unit itself, and the results are shown in table 4 . Of the 61 participants, 54% had otologic complaints related to deafness, and the data are presented in table 5; the correlation between morbidity and discontinuity of use for the hearing aid is shown in table 6 . Note: the number of complaints is greater than the number of respondents because some individuals had more than one complaint associated with hearing loss . Using the difference of proportions test, there was significant difference in the two genders for itching (p = 0.0085) and tinnitus (p = 0.0015). Note: the number of citations is greater than the number of respondents because some individuals had more than one associated morbidity . All 61 participants had hearing loss, and of these, 80.3% reported having systemic diseases, among which the most commonly reported were hypertension, hypothyroidism, hypercholesterolemia, and diabetes . The study population had an average age of 69 years for men and 72 years for women; all were considered seniors . Although the appearance of systemic diseases and hearing loss is natural, it can often be disabling and contribute negatively to quality of life, generating increasing demands for specialized health care services . To improve auditory perception, the elderly population has sought out hearing health services in sus for evaluation of hearing and fitting of hearing aids.27 28 in the present study, conducted within a hearing health clinic, most of the sample was elderly . A high incidence of morbidity was assessed in the sample, predominantly hypercholesterolemia in women and hypertension in men . Investments are necessary so that medical care for hearing health services is not restricted to ent care . According to a published study,8 the body of an elderly person has peculiarities that must be evaluated when dealing with issues related to ent disorders . The physician should consider underlying diseases, medical history, and the drugs taken by patients . In the study group, 62% of women reported having no life partner; only 19% of men did not have a partner . This coincides with work that notes the feminization of old age associated with greater longevity and independence of women compared with men.11 there was a balance between the two genders with regard to occupation, as most were retired or pensioners . A study with 320 subjects in curitiba revealed that more than half of the sample was made up of retirees and had a minimum wage income, which explains the use of the free, government - sponsored sus.27 this study (table 1) was made up of patients with predominantly sensorineural hearing loss, from mild to moderate degree with a downward sloping audiometric curve, significant for both groups . As most subjects were elderly, the prevalence of hearing loss caused by aging (i.e., presbycusis) as found in other studies had been expected.12 20 29 the majority of the sample (table 3) had been wearing a hearing aid for over 2 years, and 14 subjects (22%) reported not using the device continuously, either because of otologic factors or functional factors with the device . There was no significant difference between the two genders regarding continuous or nonuse of the device . Studies point to the fact that it is common for users to give up on wearing hearing aids for several reasons, ranging from discomfort with the sound amplification to aesthetic considerations.28 29 in this study most respondents used bte hearing aids . Of those who discontinued use of the device, half did so because of problems with the ear itself and half had problems with the device . Ear pain and itching may compromise the use of the device, because they prevent the adaptation of molds,28 and failures in the devices are among the most common causes of abandonment of use.27 hearing loss is commonly accompanied by symptoms such as tinnitus, vertigo, and aural fullness.1 the most common otologic complaint in both groups was tinnitus, reported by 48 subjects, and the second most common was vertigo, reported by 17 subjects . Tinnitus is an otologic problem that manifests itself frequently in patients with hearing loss.19 20 this problem, besides compromising the proper use of the device, may reduce its benefits and cause significant emotional and psychiatric sequelae . Vertigo also compromises the user's adequate adaptation for the hearing aid.21 contrary to the published literature18, tinnitus was significant among women in this study.18 improvement in hearing symptoms was achieved, particularly for those complaining of tinnitus (16 women and 10 men), which confirms data from the literature.20 22 this information reinforces the thesis that the hearing aid constitutes an important resource for physicians and audiologists who engage in hearing rehabilitation . After investigating the causes of otologic symptoms, systemic diseases should be studied as they may be generating their own complaints and cause discontinuation of hearing aid use because of the important relationship between hearing, metabolic, and vascular issues.3 13 15 in this study, 14 respondents who do not wear the device appropriately, referring to otologic problems such as tinnitus, vertigo, ear pain and itching, mentioned the presence of comorbidities (table 2). The literature also reports that hypertension has been shown to be a common case in both genders, but in this study a predominance of males had this complaint.10 12 finally, as reported in the literature,20 22 use of the hearing aid was decisive for reducing some complaints, such as decreased sensation of dizziness and tinnitus (table 3) for both women and men . This study showed that the incidence of systemic and otologic complaints is high in this population . During the data collection period, 278 people had follow - up visits; of these subjects, 61 (21%) reported otologic problems or operational problems with their devices . Basocochlear hearing loss, a characteristic of presbycusis, was the most prevalent both in men and women, with the majority of the subjects studied being senior citizens . The most frequently reported comorbidities were hypercholesterolemia (more significant in women) and hypertension (more significant in men). In 14 subjects, use of the device had been discontinued and there was no significant difference between genders . The reasons for discontinuation of use were itching and tinnitus, with women complaining more often . It is clear that this group of patients should be evaluated in its entirety, for actions of this nature can contribute to improving the quality of life and assisting in the process of hearing aid adaptation.
The indication for tricuspid valve (tv) surgery is mostly considered at the time of mitral or aortic valve surgery and most often because of tricuspid regurgitation (tr). Concomitant tv repair in this scenario does not significantly increase cross - clamping time and may reduce the morbidity associated with reoperation for isolated tr, leading to an increase in operative correction procedures over the past decade . Hence most follow - up studies are concerned with comparing the difference between tricuspid annuloplasty / repair (tap) and tricuspid valve replacement (tvr). However, few studies have investigated the outcome in patients with the need of tricuspid surgery because of isolated tv pathology . The etiologies here for consist mostly for annular dilation, infective endocarditis (ie), congenital (structural) pathology of the leaflets, post - interventional destruction (e.g., post - biopsy or pacemaker implantation), and rarely rheumatic disorder . Severe tr is associated with poor prognosis and surgical treatment is known to provide a superior outcome than conservative medical treatment . We present our follow - up data to evaluate long - term outcomes in patients undergoing isolated tap or tvr . Data analysis was handled anonymously and without additional patient contact or examinations, hence our institutional review board waived the need of informed consent . Between february 1995 and june 2011, 109 consecutive patients underwent isolated tv procedures at university hospital of heidelberg: 41 (37.6%) received tricuspid valve annuloplasty / repair (tap) and 68 (62.3%) underwent tricuspid valve replacement (tvr). The majority of patients suffered from tricuspid regurgitation due to structural pathology, annular dilation, or infective endocarditis . Detailed breakdown of pathologies revealed 32 patients suffered from isolated tricuspid endocarditis while 16 patients had prior pacemaker implantation causing valve defects in form of microbial vegetation, structural damage, or stenosis, 37 patients had tricuspid insufficiency (ti) because of annular dilation caused by right ventricular dilation or dcmp, while four patients had prior aortic or mitral valve replacements and developed severe ti in the further clinical course, whereas six patients suffered ti after cardiac transplantation . Six patients had congenital anomalies such as ebstein s malformation, rvoto, or asd - ii . Furthermore, four patients suffered from mechanical tk - prosthesis thrombosis, four patients had primary tricuspid stenosis and one patient developed severe insufficiency after blunt trauma causing contusio cordis . Comprehensive data such as patient demographics, cardiovascular risk factors, cardiac function assessed by two - dimensional echocardiograms, intraoperative characteristics as well as the postoperative outcomes including long - term survival were compared (tables 13). In most cases the procedures were performed through median sternotomy using cardio - pulmonary bypass (cpb) with cardioplegic arrest and standard cannulation of ascending aorta and venae cava superior and inferior (bicaval cannulation). However, some cases were performed through a transversal (n=1, 2%), parasternal (n=1, 2%), or antero - lateral (n=3, 7%) access . An alternative cannulation strategy through the femoral vessels was used in three patients (7%). There were several techniques applied depending on the morphological abnormalities of the valve including standard ring annuloplasties (n=11), ring reconstructions or tightening using the devega technique (n=6), valvuloplasty with pericardial patching or bicuscpidalization (n=20), commissurotomy (n=2), papillary muscle or chordae plasty or combination of them . The procedures were performed through median sternotomy except for two cases (3%) where the anterolateral access was used . All procedures were done under cpb with cardioplegic arrest and ascending aorta cannulation as well as bicaval cannulation . An alternative cannulation strategy through the femoral vessels was used in seven patients (10%). The choice of prosthesis was primarily based on patients age, hence patients up to the sixth decade received mechanical and patients in their sevenths decade received biological prosthesis . Exceptions were decided in respect to patients comorbidities, such as contraindications for continued anticoagulation and prosthesis were chosen in consent with the patient . Intravenous heparin infusion was started on the first postoperative day . A target activated partial thromboplastin time of 5070 seconds after removal of chest drains, warfarin medication was initiated to keep the international normalized ratio (inr) between 2.53.5 . Patients receiving biological prosthesis or annuloplasty / repair discontinued warfarin three months after surgery, if no other reason for anticoagulation existed . Patients with mechanical prosthesis needed to continue lifelong anticoagulation . All data were processed with the statistical package for social sciences (spss inc ., continuous data were shown as mean standard deviation and analyzed with the student t - test . Data analysis was handled anonymously and without additional patient contact or examinations, hence our institutional review board waived the need of informed consent . Between february 1995 and june 2011, 109 consecutive patients underwent isolated tv procedures at university hospital of heidelberg: 41 (37.6%) received tricuspid valve annuloplasty / repair (tap) and 68 (62.3%) underwent tricuspid valve replacement (tvr). The majority of patients suffered from tricuspid regurgitation due to structural pathology, annular dilation, or infective endocarditis . Detailed breakdown of pathologies revealed 32 patients suffered from isolated tricuspid endocarditis while 16 patients had prior pacemaker implantation causing valve defects in form of microbial vegetation, structural damage, or stenosis, 37 patients had tricuspid insufficiency (ti) because of annular dilation caused by right ventricular dilation or dcmp, while four patients had prior aortic or mitral valve replacements and developed severe ti in the further clinical course, whereas six patients suffered ti after cardiac transplantation . Six patients had congenital anomalies such as ebstein s malformation, rvoto, or asd - ii . Furthermore, four patients suffered from mechanical tk - prosthesis thrombosis, four patients had primary tricuspid stenosis and one patient developed severe insufficiency after blunt trauma causing contusio cordis . Comprehensive data such as patient demographics, cardiovascular risk factors, cardiac function assessed by two - dimensional echocardiograms, intraoperative characteristics as well as the postoperative outcomes including long - term survival were compared (tables 13). In most cases the procedures were performed through median sternotomy using cardio - pulmonary bypass (cpb) with cardioplegic arrest and standard cannulation of ascending aorta and venae cava superior and inferior (bicaval cannulation). However, some cases were performed through a transversal (n=1, 2%), parasternal (n=1, 2%), or antero - lateral (n=3, 7%) access . An alternative cannulation strategy through the femoral vessels was used in three patients (7%). There were several techniques applied depending on the morphological abnormalities of the valve including standard ring annuloplasties (n=11), ring reconstructions or tightening using the devega technique (n=6), valvuloplasty with pericardial patching or bicuscpidalization (n=20), commissurotomy (n=2), papillary muscle or chordae plasty or combination of them . In most cases the procedures were performed through median sternotomy except for two cases (3%) where the anterolateral access was used . All procedures were done under cpb with cardioplegic arrest and ascending aorta cannulation as well as bicaval cannulation . An alternative cannulation strategy through the femoral vessels was used in seven patients (10%). The choice of prosthesis was primarily based on patients age, hence patients up to the sixth decade received mechanical and patients in their sevenths decade received biological prosthesis . Exceptions were decided in respect to patients comorbidities, such as contraindications for continued anticoagulation and prosthesis were chosen in consent with the patient . Intravenous heparin infusion was started on the first postoperative day . A target activated partial thromboplastin time of 5070 seconds after removal of chest drains, warfarin medication was initiated to keep the international normalized ratio (inr) between 2.53.5 . Patients receiving biological prosthesis or annuloplasty / repair discontinued warfarin three months after surgery, if no other reason for anticoagulation existed . Patients with mechanical prosthesis needed to continue lifelong anticoagulation . All data were processed with the statistical package for social sciences (spss inc ., continuous data were shown as mean standard deviation and analyzed with the student t - test . There were no statistically significant differences between the two groups regarding age (50.719.4 years in the tap group vs. 55.715.9 years in the tvr group, p=0.163), gender distribution (43.9% vs. 54.4% female patients in the tap and tvr groups, respectively, p=0.305), and body mass index (bmi) (24.55.2 in the tap group vs. 24.96.9 in the tvr group, p=0.713). Moreover, there were no significant differences regarding preoperative angina or dyspnea status, liver, renal and pulmonary function, infections, hemodynamic status, and cardiovascular risk factors (table 1). Furthermore, the differences between the two groups in terms of previous coronary (p=0.481), aortic valve (p=1.000), mitral valve (p=0.530), or pulmonary valve surgery (p=1.000) was not statistically significant, whereas patients in the tvr group had a significantly higher rate of previous tricuspid valve repair (p=0.013). Preoperative cardiac function assessed by transesophageal echocardiography was also similar and the severity of the disease reflected by pulmonary hypertension and right ventricular function did not differ significantly in both groups (table 2). Additionally, there was no significant difference in the intraoperative characteristics in terms of cross clamping time or urgency of the procedure (table 3). Postoperatively, patients from both groups had similar left ventricular and right ventricular function assessed by two - dimensional echocardiography . There were no statistically significant differences in terms of postoperative arrhythmias, the need for intra - aortic balloon pump or ventricular assist device implantations . Both groups were comparable in terms of inotropic support requirement, postoperatively, except for noradrenaline support which was needed more frequently in the replacement group (50.0% in the tvr group vs. 24.4% in the tap group, p=0.014). The distribution of postoperative complications such as renal failure requiring conservative treatment, dialysis / hemofiltration, pleural effusions requiring negative balance, or drainage was similar in both groups (table 4). Early survival at 30 days after surgery was 97.6% in the tap group and 91.1% in the tvr group . After 6 months, 89.1% in the tap group and 87.8% in the tvr group were alive . In terms of long - term survival, there was no further mortality observed after one year post - surgery in both groups (log rank p=0.919, breslow p=0.834, tarone - ware p=0.880) in the kaplan - meier survival analysis . The 1-, 5-, and 8-year survival rates were 85.8% for tap and 87.8% for tvr group (figure 1). Significant tricuspid disease requiring surgical correction is a challenging pathology, demanding critical decision - making regarding reconstruction versus replacement and timing of the procedure . The reasons here for are the reported high mortality and the fact that tr is relatively well tolerated, even if severe . Hence patients present with complex morbidity such as manifest congestive heart failure, severe cardiac dilation, pulmonary hypertension or endocarditis . The presence of these serious preoperative conditions present highly confounding factors altering postoperative outcomes significantly and probably accounting for previously reported poor outcomes . Currently the indication for surgical correction vs. replacement remains at the surgeon s discretion, although recently there is a growing tendency towards reconstructive strategies . Theoretical benefits are the avoidance of inserting a rigid prosthesis into the thin - walled, low - pressure right ventricle, which can result further deterioration of right - ventricular dysfunction [911]. However, early and long - term outcomes after tap show high rates of recurrent tr despite the use of annuloplasty rings . In a large series reported by the toronto group and the cleveland clinic group the recurrence of moderate to this can account for diminished survival because deteriorating right ventricular function and redo tricuspid valve surgery in that case is associated with higher morbidity and mortality . However, most studies include a variety of patients in their analysis, especially those in need of multiple cardiac interventions . In this study however, only isolated tricuspid valve pathologies were analyzed, thus our patient cohort was more uniform concerning preoperative characteristics and direct comparison between the test groups is feasible without the need for statistical alterations such as pair - matched analysis or propensity score matching . Age, gender, bmi, and the severity of the disease were comparable in both groups . Known preoperative risk factors like right ventricular function and pulmonary hypertension were also similar, as well as known operative confounding factors like cross clamping time or the urgency of the procedure . The overall results of this study show that the outcome after tricuspid surgery is acceptable with 30-day survival of 97.6% in the tap group and 91.1% in the tvr group and long - term survival of 85.8% in the tap and 87.8% in the tvr group . These rates are in accordance with, if not better, than in previous published studies . However, our promising results might be overestimated by a relatively small number of patients and therefore lower statistical power . Nonetheless the outcomes are somewhat disappointing when compared with survival after left sided valve replacement operations, even after exclusion of patients with complex cardiac pathologies needing multiple surgical corrections . With survival rates at 1-, 5-, and 10 years of 80%, 72%, and 66% for repair and 85%, 79%, and 49% respectively for the replacement group, without statistical significance . The incidence of major adverse cardiac events was also similar in both groups, as were clinical surrogate parameters, except for the use of noradrenaline: which was significantly more needed in the tricuspid replacement group . This may be due to the fact that this patient group had a longer cpb time and thus suffered more vasoplegia in the early postoperative period . Thus the main finding of this study is that there is no clear benefit when comparing tricuspid repair to replacement . Hence the growing enthusiasm towards corrective procedures needs to be put into perspective and focus needs to be shifted towards the timing of the procedure to maintain right ventricular function . Current guidelines recommend surgical intervention when patients become symptomatic, which might lead to significant progression of the disease and further deterioration of right ventricular function . However, it has been shown that tr reduces exercise capacity and negatively affects long - term outcome, irrespective of pulmonary hypertension or left ventricular function [1619]. Echocardiographic parameters of right ventricular function could be consulted when determining optimal timing of surgical intervention . The decision should lean towards valve replacement in patients with reasonable suspicion of recurrent regurgitation.
Methicillin - resistant staphylococcus aureus (mrsa) is recognized as one of the most common pathogens of both nosocomial and community - acquired infections worldwide . As a feature distinct from methicillin - susceptible s. aureus (mssa), mrsa has a transmissible genome element, staphylococcal cassette chromosome mec (sccmec), inserted in a specific site of the chromosome . The sccmec in mrsa has been differentiated into at least 12 genetic types (i xii),, among which types i iii have been traditionally associated with hospital - acquired mrsa (ha - mrsa), while type iv and v have been commonly found in community - acquired mrsa (ca - mrsa). However, in recent years, ca - mrsa with the dominant sccmec types (iv and v) has been brought to healthcare settings causing nosocomial infections,,, which makes distinction between ha- and ca - mrsa more difficult in terms of sccmec type . The pathogenesis of many ca - mrsa strains have been attributed to the production of panton - valentine leukocidin (pvl), a two - component toxin encoded by two genes, lukf - pv and luk - s - pv, which are carried on lysogenic bacteriophages, . Accordingly, pvl - positive s. aureus is associated with severe symptoms in a wide spectrum of infections including skin and soft tissue infections and necrotizing pneumonia, . Prevalence of ca - mrsa harbouring pvl genes has been increasing recently in hospitalized patients as well as healthy individuals in the community, . Distribution and spread of mrsa clones on a global scale have been revealed by genetic classifications with multilocus sequence typing and sccmec typing, . Several ha - mrsa clones including st5-mrsa - sccmec ii (st5-ii, ny / japan clone) and st22-iv (emrsa-15) are known as pandemic clones predominating in east asia / north america and europe, respectively . In contrast, various clones have been documented for ca - mrsa which are distributed locally or predominate in a region, often associated with international spread . Globally predominant ca - mrsa includes five clones, i.e. St1 (usa400 clone), st8 (usa300 clone), st30 (south west pacific clone), st59 (taiwan clone) and st80 (european clone), among which st8 and st30 are considered pandemic as a result of its distribution to every continent . In asia, two pandemic ha - mrsa clones with st5 and st239 are disseminating, whereas various ca - mrsa clones including those with st8, st30, st59, st72 and st772 have been reported . In nepal, the prevalence of mrsa from clinical specimens in hospitals has been described to be 2669% in several studies via antimicrobial susceptibility testing,,,,, although the rate varies depending on the types of infections or specimens examined . A recent study revealed a high prevalence of pvl genes in nosocomial isolates of mrsa and mssa (26% and 52% respectively). However, in nepal, there have been no studies conducted on genotypes (st and sccmec types) of clinical mrsa isolates, particularly pvl - positive isolates . We found high prevalence of pvl in mrsa and mssa, as well as the presence of pvl - positive st772 mrsa - v (bengal bay clone). A deletion variant of elastin binding protein gene was first identified in st22 s. aureus isolates and its origin was analysed . From august 2012 to october 2012, about 200 s. aureus isolates were collected from two general hospitals (approximately 100 isolates each) with more than 500 beds in kathmandu, nepal . The main specimen of the isolates was pus (n = 84), followed by urine (n = 12), sputum and blood (n = 2 each). A single isolate from each individual patient was subjected to study . Bacterial isolation and species identification were performed by conventional microbiological methods, and the presence of nuclease gene was confirmed by multiplex pcr . Individual bacterial strains were stored in microbank (pro - lab diagnostics, richmond hill, on, canada) at 80c and recovered when they were analysed . Staphylococcal 16s rrna, nuc, meca, pvl gene (luks - pv / lukf - pv) and acme - arca (arginine deiminase gene) were detected for all isolates by multiplex pcr assay as described by zhang et al . . Minimum inhibitory concentrations against 18 antimicrobial agents based on the broth microdilution test were measured by using dry plate eiken dp32 (eiken chemical, tokyo, japan). Breakpoints defined in the clinical laboratory standards institute guidelines were used to distinguish between resistant and susceptible strains for most of drugs examined . Staphylocoagulase genotype (coa type) of s. aureus isolates was determined by multiplex pcr using previously published primers and conditions . For the strains for which the coa types were not determined for i x by the multiplex pcr, partial coa sequences (d1, d2 and the central regions) were determined as described previously, to assign the coa genotype by sequence identity via blast search (http://blast.ncbi.nlm.nih.gov/blast.cgi). For selected isolates, sequence type (st) was determined according to the scheme of multilocus sequence typing, and agr group was classified as described previously . Presence of genes encoding enterotoxins and other toxins, adhesins, other proteins related to virulence and antimicrobial resistance genes was analysed by multiplex or uniplex pcr using primers described previously, . The sequence of the gene encoding elastin binding protein (ebps) was determined by pcr and direct sequencing, as described previously . Multiple alignment of ebps sequences determined in the present study and those retrieved from the genbank database was performed by the multalin interface (http://multalin.toulouse.inra.fr/multalin/). The lalign program (european bioinformatics institute; http://www.ebi.ac.uk/tools/psa/lalign/nucleotide.html) a phylogenetic tree of ebps was constructed by the neighbour - joining method by mega 5.01 software, statistically supported by bootstrapping with 1000 replicates . Full - length ebps sequences of strains np173, np177 and np199 determined in the present study were deposited in the genbank database under accession numbers kt951674kt951676 respectively . Among the 100 s. aureus clinical isolates examined, 32 isolates were mrsa which had sccmec type v (n = 20, 62.5%) or type iv (n = 2, 6.3%) (table 1), while sccmec type was not identified for ten isolates . Pvl genes were detected in 78% (25/32) of mrsa and 71% (48/68) of mssa isolates . The most common coagulase genotypes of pvl - positive mssa and mrsa were iva and via, respectively . Genetic characteristics were analysed for 17 isolates as representatives of pvl - positive mrsa (seven isolates), pvl - positive mssa (six isolates), pvl - negative mrsa (three isolates) and a pvl - negative mssa isolate (table 2). The pvl - positive mrsa isolates belonged to st1, st22, st88 or st772 . The st772 was identified into mrsa with sccmec v, coa - via and agr - ii (one pvl - negative and four pvl - positive isolates), as well as two pvl - positive mssa . Mrsa isolates with or without pvl were mostly resistant to ampicillin, gentamicin and levofloxacin, and had generally more drug resistance genes than pvl - positive mssa, although some meca - positive isolates (mrsa) were susceptible to oxacillin (supplementary table s1). Although luke - lukd and haemolysin genes were detected in most isolates examined, st772 isolates (mrsa and mssa) and st22 mrsa had more enterotoxin genes than st1 and st88 mrsa and st30 mssa isolates (table 2). In the present study, we first demonstrated the presence of st772-mrsa - v and st22-mrsa - iv in nepal . St772 and st22 have been reported as epidemic clones associated with infections in both community and healthcare settings in india,,, . St772 mssa was originally reported in bangladesh; thereafter, st772-mrsa - v was identified in india, followed by transmission to east / southeast asia, australia, new zealand, the middle east and europe . This clone is colloquially referred to as the bengal bay clone, and it is mostly pvl positive and relatively multiresistant compared to other ca - mrsa . In addition to the increasing prevalence of st772-mrsa - v in india, detection of this clone outside india has been related to travel history to or from india,, . Because of its adjacent location, it is conceivable that st772-mrsa - v in india might have been readily transmitted to nepal, or it may have been originally endemic in nepal as well as india . Although the elastin binding protein (ebps) gene (ebps) was detected in all the isolates examined by pcr with primers described previously, pcr products that were shorter than the expected size (652 bp) were found in four st22 isolates (data not shown), among which three isolates (np173, np177 and np199; pvl - positive mrsa, pvl - negative mrsa and pvl - positive mssa respectively) were further analysed for their ebps gene sequences . These ebps genes were revealed to be a variant (ebps - v) with an internal deletion of 180 bp encoding a 60 aa sequence . By blast search, sequences similar to ebps - v were identified in strain 71a_s11 (st22), 93b_s9 and y12 (st121) and 6850 (st50). Alignment of the deduced amino acid sequences of the ebps - v identified in the present study with those of other representative s. aureus strains is shown in fig . 1 . The deleted portion (60 aa) of ebps - v in st22 nepalese strains, corresponding to aa 199258 of ebps from strain col, was identical to those found in strains 71a_s11, 93b_s9, y12 and 6850 (genbank accession nos . Phylogenetic analysis of the ebps from various s. aureus strains, including ebps - v, revealed the presence of three major lineages (i, ii and iii) (fig . 2). Lineage ii contained the st22 cluster, which consisted of ebps - v from nepalese strains and intact ebps in st22 strains as emrsa-15 . Nucleotide sequence identity of intact ebps within the same lineage was more than 98.8%, while it was 95.298% between different lineages (supplementary table s2). Ebps - v of st22 nepalese strains showed> 99% identity with each other, but slightly lower identity was found in st121 strains (95.695.7%). The variant of ebps with a 180 bp deletion was first reported for isolates from orthopaedic infections in italy, although their st was not identified . Thereafter we identified a similar ebps variant (ebps - v) in st121 mssa isolates in myanmar as well as in st121 isolates in bangladesh and japan . The present study elucidated that ebps - v of st22 and st121 belong to different lineages, suggesting that the 180 bp deletion event in ebps might have occurred in st22 s. aureus and st121 s. aureus independently . Ebps, one of the adhesins that binds to host cellular matrix factors involved in biofilm formation, is produced by most mrsa examined so far,, . Ebps is a cell - surface molecule and mediates binding of bacterial cell to soluble elastin peptides and tropoelastin,, with its n - terminal region (aa 1434) a ligand - binding domain exposed on the surface of the cell and two (h1 and h3) among the three putative hydrophobic domains spanning the cell membrane . Although the functional and structural changes caused by the deletion in st22 isolates are unknown, the n - terminal region must be exposed on the surface of cell for the normal function of ebps - v; accordingly, the h2 region is necessary to span the membrane instead of the deleted h1 region (fig . 1, supplementary fig . S1). In that case, only the form of the cytoplasmic portion of ebps - v may be different from that of ebps, which may be possible to cause any functional difference . It was notable in the present study that all the st22 s. aureus, including mrsa and mssa, harboured ebps - v, while intact ebps was also found in other previously reported st22 strains including emrsa-15 . In contrast, all the st121 s. aureus analysed to date harboured ebps - v,, . The st121 clone is known to be a common cause of skin and soft tissue infection disseminating globally, mostly meca negative but pvl positive, exhibiting higher virulence . Although the association of ebps - v with the enhanced virulence of the st121 clone is not known, if the presence of ebps - v affects phenotypic trait of st22 s. aureus (e.g. Virulence or fitness advantage to environment), it is possible that the proportions of st22 isolates with ebps and ebps - v may change over time epidemiologically . Further studies thus may be necessary to monitor the prevalence of st22 s. aureus with ebps - v.
Chronic obstructive pulmonary disease (copd) is a progressive obstructive airways disease with incomplete improvement in lung function in response to therapy . Copd is estimated to affect 10% of the world s population aged 40 years and its prevalence is expected to continue to increase over the next decade or more.1 an accelerated decline in forced expiratory volume in 1 second (fev1) occurs alongside a decline in ability to undertake physical activities and social functioning, reflected in the measurement of clinical outcomes including dyspnea and health status.2 regular pharmacotherapy using long - acting inhaled bronchodilators has been shown to provide not only rapid improvement in lung function, but clinically important decreases in dyspnea and exacerbation rate with a corresponding improvement in health status.3 bronchodilators have therefore become the cornerstone of treatment for copd of all severity stages . Current guidelines recommend that patients with moderate to severe copd initiate therapy with long - acting bronchodilators if short - acting bronchodilators are not providing sufficient symptom relief.4 currently available agents include the twice - daily long - acting 2-agonists (labas) formoterol and salmeterol and the once - daily anticholinergic tiotropium . Although long - acting bronchodilators are considered more effective and convenient than short - acting agents,5 symptoms remain limiting for many subjects.6 the shift in treatment preference from short - acting bronchodilators with multiple dosing per day to long - acting bronchodilators with once - daily or twice - daily dosing and prolonged duration of bronchodilation has resulted in improved clinical outcomes for copd patients.7 the consequent reduction in dosing frequency not only simplifies disease management, but has the potential to improve patient adherence and compliance.5 until recently, the only available labas were salmeterol and formoterol, both of which have an approximate 12-hour duration of bronchodilator action, and hence are used twice daily for maintenance therapy in copd . Indacaterol is the first once - daily long - acting 2-selective agent, sometimes referred to as an ultra - laba, indicated for maintenance treatment in patients with moderate to severe copd, has been approved in more than 40 countries (including throughout the european union) at a recommended dose of 150 g once daily and a maximum dose of 300 g once daily,8 and has been shown to be effective and well tolerated by subjects with copd during 12 weeks of treatment.9 with any new treatment intended for long - term use, it is important to evaluate safety and to determine if efficacy remains unblunted with regular use . A thorough characterization of safety is particularly relevant for a long - acting bronchodilator, given the recent interest in the safety of these agents in the treatment of copd.10 the 75 g dose of indacaterol as licensed in the us was evaluated only in studies of 12 weeks duration.11 overall, the results with indacaterol 150 g and 300 g demonstrate a good profile of safety and tolerability that is comparable with the other evaluated long - acting bronchodilators . Compared with these two doses, results with the 75 g dose of indacaterol were occasionally anomalous but must be considered in the context that these data are less robust in terms of shorter length of treatment (maximum 12 weeks) and fewer patients exposed to this dose.12 however, in the two studies evaluating the 75 g dose, the safety and tolerability profiles were similar to placebo.11 few deaths occurred in any of the active treatment groups, and the number of deaths adjusted per patient - year was not increased with any of the laba treatments relative to placebo . The finding of no deaths among the indacaterol 75 g group should be interpreted with caution, given that most of the data came from two 12-week studies.12 sequential modifications of the chemical structure of catecholamines13 allowed synthesis of agonists with improved selectivity for the 2-adrenoreceptor and led to the subsequent development of short - acting 2-bronchodilators such as fenoterol, salbutamol (albuterol), and terbutaline, and the long - acting 2-agonists, salmeterol and formoterol . In vitro studies during the preclinical development of indacaterol13 characterized this new agent as having high agonist efficacy at the human 2-adrenoreceptor, with a binding affinity similar to formoterol, an intrinsic activity higher than salmeterol, and a functional selectivity similar to formoterol over the 1-adrenoreceptor, and similar to formoterol and salbutamol over the 3-adrenoreceptor . In vivo, indacaterol produced a prolonged bronchoprotective effect against pharmacologically induced bronchoconstriction, and showed an improved cardiovascular safety profile.14 reports on pharmacokinetics15 showed that indacaterol is rapidly absorbed and distributed after inhalation, with a median time to reach peak serum concentrations of approximately 15 minutes after single or repeated inhaled doses, although systemic exposure results from a composite of pulmonary and intestinal absorption . The faster onset and longer duration of action of indacaterol compared with some other 2-adrenoceptor agonists may be related to lipid membrane interactions . The sum of these small differences, including higher partitioning of indacaterol into the microenvironment of the receptor and its faster membrane permeation, is likely to contribute to its faster onset and longer duration of therapeutic action.16 in the toxicological assessments,17 indacaterol was considered negative in the standard battery of in vitro and in vivo genotoxicity tests, and did not raise concerns regarding potential carcinogenicity . An analysis of the effect of age, gender, and weight on systemic exposure after inhalation indicated that indacaterol can be used safely in all age and weight groups within the copd population, and regardless of gender . Pharmacokinetic data taken during multiple - dose studies of indacaterol 400 g or 800 g once daily for 14 days demonstrated rapid absorption and a mean elimination half- life> 30 hours, whereas in a single - dose study, doses between 600 g and 2000 g were rapidly absorbed, with maximum serum concentrations reached within 15 minutes.18 in large - scale registration studies, indacaterol provided 24-hour bronchodilation on once - daily dosing with an effect that was sustained during treatment for up to 1 year.19 the relative effect of indacaterol on trough fev1 (measured 24 hours following dosing) after 12 weeks of treatment was reported to be 4050 ml greater than the once - daily anticholinergic bronchodilator, tiotropium, and 60100 ml greater than the trough fev1 measured 12 hours after dosing with the twice - daily 2-agonists, salmeterol and formoterol20 (figure 1). The efficacy and safety of indacaterol was evaluated in an extensive phase iii clinical program in which patients received doses of up to 600 g once daily for up to 52 weeks.20 in an analysis of data from 801 patients with moderate to severe copd after 2 weeks of treatment, indacaterol 150 g once daily was identified as the lowest dose that was numerically superior to the active comparators (formoterol twice daily and open - label tiotropium once daily) and, along with the next highest dose (300 g), was selected for further evaluation.21 chapman et al22 recently reported on the safety of indacaterol 150 g and 300 g once daily versus placebo during the 26-week extension phase of the 26-week open - label tiotropium comparison study, thereby providing data over a total duration of 52 weeks . In their analysis, adverse events occurred in 76%, 77%, and 68% of subjects receiving indacaterol 150 g, 300 g, and placebo, respectively, whereas serious adverse events occurred in 10.4%, 12.3%, and 10.5%, respectively . There were no clinically significant effects with indacaterol at either dose on qtc interval or potassium or glucose levels . This additional evaluation showed that indacaterol 150 g and 300 g provided statistically significant and clinically relevant improvements in trough fev1 versus placebo up to 26 weeks.19 although indacaterol 150 g and 300 g had similar effects on trough fev1, the higher dose was associated with incremental benefits in terms of symptomatic relief, such as dyspnea, particularly for patients with more severe copd . Further, the overall clinical trial program indicated that indacaterol has a similar safety and tolerability profile across all of the doses evaluated.22,23 results of a recent 14-day crossover study24 that compared indacaterol 300 g with open - label salmeterol showed that indacaterol dosed once daily has a bronchodilator profile that is consistently numerically superior to salmeterol dosed twice daily . A larger 26-week comparison with blinded salmeterol suggested that in addition to improved bronchodilator efficacy, once - daily dosing with indacaterol is generally more effective than twice - daily salmeterol.23 copd exacerbations were significantly reduced versus placebo for indacaterol 150 g or 300 g once daily . In a 52-week study,20 once - daily treatment with indacaterol prolonged the time to first copd exacerbation and was effective in reducing the incidence and frequency of copd exacerbations, with no significant difference between indacaterol and formoterol . Patients treated with indacaterol had a significantly higher percentage of days with no use of as - needed rescue salbutamol than placebo recipients in all large studies . Only the once - daily bronchodilators (indacaterol and tiotropium) provided more than the 120 ml improvement in trough fev1, prespecified as the threshold of clinical interest in several studies studies.9,19,20,23 for trough fev1 at week 12, indacaterol 150 g and 300 g were statistically superior to open - label tiotropium, indacaterol 150 g was statistically superior to salmeterol, and indacaterol 300 g was statistically superior to formoterol . The efficacy of indacaterol was maintained over the study durations (ie, 6 months19,23 and 1 year20). The mean fev1 measured at 5 minutes after the first dose with indacaterol was 110130 ml greater than after placebo (p <0.001), approximately double the corresponding values with tiotropium (70 ml; p <0.001 versus tiotropium) and salmeterol (60 ml; p <0.001 versus salmeterol), and similar to formoterol (140 ml; not significantly different, figure 2). Indacaterol improved breathlessness assessed using the transition dyspnea index, with an effect close to or greater than the threshold for clinical relevance . Indacaterol had a similar (150 g dose) or greater (300 g dose) effect than open - label tiotropium, and a greater effect than the twice - daily bronchodilators formoterol and salmeterol . These differences are evident both in terms of the effect on mean transition dyspnea index scores and the percentage of patients who achieved at least a clinically relevant improvement in breathlessness . Indacaterol, along with all the other bronchodilators, showed a statistically significant effect compared with placebo at later time points during the 6-month and 1-year studies.9,19,20,23 patients with copd treated with indacaterol used less as - needed salbutamol, with a decrease of approximately 1.5 puffs per day from baseline . Patients receiving indacaterol also recorded more days when they took no salbutamol (55%60%), which is more than was recorded with tiotropium (46%) and formoterol (52%).9,19,20,23 exacerbation rates were numerically reduced compared with placebo with all the bronchodilators, but in most cases the effect was not statistically significant . These studies were not designed to measure exacerbations as a primary outcome, so only stable patients were recruited (annual exacerbation rates with placebo treatment were in the range 0.71.0).9,19,20,23 health status, assessed using the st george s respiratory questionnaire, was improved relative to placebo with indacaterol and with the two twice - daily bronchodilators, but not with tiotropium, although it should be noted that tiotropium was given as seen in previous studies with bronchodilators, most treatments did not produce a change that exceeded the minimal clinically important difference when compared with placebo, but this was achieved with indacaterol 150 g9,23 and with salmeterol.23 a similar pattern of results was seen for patients with severe copd (global initiative for chronic obstructive lung disease [gold] stage iii). This is appropriate for primary care, given that a recent large european study in primary care found that nearly 40% of patients had gold stage iii or iv disease.25 systemic bioavailability of 2-agonist bronchodilators can elicit known 2-adrenoceptor - mediated cardiovascular effects,26 such as palpitations, tachycardia, changes in blood pressure, and electrocardiographic abnormalities such as a prolonged qt interval, as well as hypokalemia, hyperglycemia, headache, and skeletal muscle tremor . The available data for indacaterol overall support an excellent safety profile, with no relevance of its long duration of action in terms of side effects . The bronchodilator efficacy of indacaterol increased from the first dose to the measurements performed at the end of 12 weeks of treatment . In a longer - term study, the efficacy of indacaterol 150 g once daily was not reduced with repeated dosing for 6 or 12 months, showing persistence of bronchodilator response without tachyphylaxis.22 in copd patients, a large data set was generated from various studies primarily aimed at dose ranging and safety analysis.9,27 safety variables were also secondary endpoints in all studies primarily aimed at efficacy endpoints . Overall, the rate of adverse events was low and similar across treatment groups in the different trials . There was no evidence of drug - related or dose - related changes in hematological parameters, and no clinically relevant differences were observed between treatment groups in any of the biochemical variables measured . The incidence of other 2-agonist - related effects, including muscle spasm, headache, and tremor, was comparable between indacaterol and placebo groups . Data generated across a wide dose range support a good cardiovascular safety, with no clinically significant differences in mean pulse rate, no drug - related trends in systolic or diastolic blood pressure, no statistically significant differences in mean qtc interval, and no differences in the numbers of subjects with notable qtc interval increases . Serious adverse events in subjects receiving indacaterol occurred with frequencies similar or inferior to placebo groups, and none were classified as suspected to be related to the study drug in copd patients in the trials reported . In various trials, cough upon drug inhalation was reported with a mean frequency ranging from 2.9% to 17.8% in indacaterol groups and 0.9% to 7.3% in placebo groups . The cough was described as occurring within 15 seconds following drug inhalation, with a median duration of 6 seconds, and having no association with bronchospasm or increased study discontinuation rates.9 more than 4000 patients have completed treatment with indacaterol so far in controlled clinical studies of at least 12 weeks duration, and no significant safety concerns have arisen . Safety has been evaluated during up to 1 year s treatment with the approved daily doses of 150 g and 300 g, and with a higher (unlicensed) dose of 600 g once daily . The most common adverse events reflected the symptoms and manifestations of copd, such as worsening of copd and respiratory tract infections . As with the other long - acting bronchodilators,28 indacaterol has a good profile of cardiovascular safety in patients with copd, and has little or no effect on vital signs and qtc interval, the derived electrocardiographic measurement used to indicate risk of arrhythmias.29 approximately 20% of patients experience a mild transient cough in the first few minutes after inhalation of indacaterol . This typically lasts for several seconds and is not associated with loss of efficacy, increased dropout rates, or any safety concerns . The dosing regimen requiring less frequent dosing was shown to be associated with improved patient compliance with therapy.30 therefore, an effective once - daily 2-agonist bronchodilator would add a significant advance to the therapeutic toolbox for copd . Long - term safety of a new treatment is important for subjects with copd, who are often elderly, frequently have significant comorbidity, and tend to be receiving multiple medications.31 in all studies designed to investigate whether indacaterol has the same tolerability as that of the labas already on the market, indacaterol was well tolerated at all doses and with a good overall safety profile.32 the inhalation device used for indacaterol, ie, the breezhaler, is a simple dry powder inhaler in which capsules of powdered medication are loaded, pierced, and inhaled . While similar in function to the handihaler used for tiotropium, the breezhaler is a lower resistance device . When compared with the handihaler in a 2-week patient handling and preference study, patients handled both devices well and without error, but preferred the smaller size and lower resistance characteristics of the breezhaler.33 indacaterol is a new once - daily laba with a rapid onset of action (within 5 minutes), a peak effect at approximately 3 hours, and a duration of bronchodilation lasting at least 24 hours . Indacaterol has become the first effective once - daily laba that is widely approved for use in copd . Indacaterol, now approved in the european union and us for copd, provides effective 24-hour bronchodilation and a fast onset of action, with an efficacy at least comparable or superior to current bronchodilator therapy standards . We restrict our discussion only to the european product because in the us there were only two studies that evaluated 12 weeks duration . The data from clinical development support a favorable safety and tolerability profile within the 2-agonist drug class, with no relevant safety issues identified . The recommended dose is 150 g once daily, delivered using a single - dose dry powder inhaler device . A 300 g per capsule presentation for once - daily dosing is also approved and may provide additional clinical benefit for patients with severe copd . As expected, improvements in lung function have translated into correspondingly beneficial effects on patient - reported outcomes, including significant reductions in exacerbations, dyspnea, and days with poor control, and clinically meaningful improvements in health - related quality of life, particularly when comparisons are made with placebo . Some evidence shows that indacaterol is suitable for use as first - line monotherapy in copd patients with moderate disease (gold stage ii) and beyond that do not require inhaled corticosteroids as per gold guidelines, or in combination with an inhaled corticosteroid in severe or very severe patients with repeated exacerbations . As with long - acting bronchodilators, a short - acting bronchodilator such as salbutamol further research should provide evidence on the benefits and safety profile of combining indacaterol with a long - acting anticholinergic bronchodilator . Current copd guidelines recommend inhaled bronchodilators as the mainstay of copd pharmacotherapy and at least one long - acting inhaled bronchodilator as first - line maintenance therapy for patients with moderate symptomatic copd, without signifying a preference for either a laba or a once - daily long - acting antimuscarinic (lama). However, in actual practice, when a single long - acting inhaled bronchodilator is prescribed, it is most often a lama (tiotropium), rather than a twice - daily laba (salmeterol or formoterol). The reason for this real world preference is unclear, but may be related to the greater convenience of the once - daily lama than the twice - daily laba, a distinction that will be eliminated when indacaterol becomes widely available . The gold guidelines also recommend combining a lama and a laba in copd patients not responding satisfactorily to monotherapy with either class of long - acting inhaled bronchodilator, a strategy that has been found to result in additive improvements in lung function in short - term clinical trials . However, this recommendation is infrequently followed; instead, in patients not responding to tiotropium alone, a laba - inhaled corticosteroid combination is generally added as the next step . The availability of a once - daily laba, such as indacaterol, would provide a convenient alternative to adding a twice - daily laba - inhaled corticosteroid to a once - daily lama because the combined use of tiotropium and indacaterol in separate devices would entail only once - daily dosing . Indacaterol provides a level of bronchodilation that is similar to tiotropium and greater than the twice - daily agents, formoterol and salmeterol . The measures of breathlessness and health status used in the indacaterol studies encompass components reflecting symptoms and the ability to undertake activities of daily living . Indacaterol was effective at reducing breathlessness, the most troublesome copd symptom, and the 300 g dose was significantly more effective in this regard than tiotropium and the twice - daily agents . The beneficial effects of indacaterol on breathlessness and health status in gold stage ii patients suggest that the overall study results provide a useful guide to the level of efficacy that may be expected in milder patients who may be seen predominantly in a primary care patient population . Indacaterol can also be combined with a once - daily inhaled corticosteroid, thus providing a more convenient once - daily alternative to existing twice - daily laba - inhaled corticosteroid combinations (fluticasone - salmeterol and budesonide - formoterol) for the treatment of severe copd . A noticeable side effect has been cough, which has been mild and with attenuation over time and without pre - mature withdrawal from the trials . It has occurred within seconds of inhalation with rapid resolution, and has not been associated with bronchospasm . The mechanism of the cough remains unclear but presumably is related to a drug - specific stimulant effect on cough receptors in the upper / central airways; it seems that tachyphylaxis to this effect develops over time . No clinically meaningful effects have been noted on cardiovascular parameters, including heart rate, blood pressure, or qtc interval, or on serum potassium or blood glucose . A patient with confirmed copd who remains troubled by symptoms limiting daily activities despite a short - acting bronchodilator is a candidate for long - acting bronchodilator treatment . Factors to consider are the patient s symptomatic response and preference, as well as drug side effects and cost . Indacaterol is attractive as a once - daily bronchodilator because it has slightly greater efficacy than the twice - daily bronchodilators . On the basis of the evidence reviewed, we conclude that once - daily indacaterol is an effective and beneficial maintenance bronchodilator treatment for patients with moderate to severe copd, including patients with gold stage ii disease . It is likely that once - daily dosing of a bronchodilator would be a significant convenience and probably a compliance - enhancing advantage, leading to improved overall clinical outcomes in patients with copd.
Both vogt - koyanagi - harada (vkh) disease and central serous chorioretinopathy (csc) develop serous retinal detachment, and since recently, it has been well known that in both diseases, the thick choroid can be seen on optical coherence tomography (oct). However, the pathogenesis of each disease is totally different; vkh is an inflammatory disease affecting melanocytes, while csc is a disorder of choroidal circulation . Steroids treat vkh but worsen csc; therefore, it is important to distinguish these diseases . We report a case of vkh disease in which recurred serous retinal detachment was treated successfully with steroids, but the last recurrence did not respond to the steroids and angiographic findings suggested csc . Here, we show the multimodal images of this case including en face oct images during the course of vkh disease . In this study, we use swept - source oct (dri oct-1, topcon, tokyo, japan), which allows superior visualization of the choroid thanks to a 1,050-nm wavelength light source and a scanning speed of 100,000 a scans / second . The axial and transverse resolution of the system was 8 and 20 m, respectively . The oct scan protocol was 3d scan covering an area of 12 9 mm horizontal and vertical b scans . The software enabled us to reconstruct a series of en face images from 3d volume scan, where 992 consecutive en face images were created with a depth interval of 2.6 m . When observing choroidal vessels, we referred to the en face images around the depth of the upper one third of haller's layer, where the choroidal vessels were seen clearly at each visit . A 50-year - old man was referred to the sumitomo hospital with blurring of vision in his right eye from 2 weeks prior . His best - corrected decimal visual acuity (bcva) on presentation was 0.8 in the right eye and 1.2 in the left eye . Intraocular pressure was normal and no remarkable inflammation was observed in either the anterior or posterior chamber in both eyes . Fundus and oct examination showed bilateral optic disc swelling and exudative subretinal fluid (srf) in his right eye . Choroid was significantly thick in both eyes: much thicker in the right eye than in the left eye (593 and 393 m, respectively; fig . En face oct images showed blurry choroidal vessels and retinal pigment epithelium (rpe) folds associated with the disc swelling bilaterally (fig . Fluorescein angiography (fa) and indocyanine green angiography (icga) was performed using spectral - domain oct (spectralis, heidelberg engineering, heidelberg, germany). Fa showed the leakage from the optic disc bilaterally and pooling of the dye in the subretinal space at the macula in the right eye (fig . 1e, f) and icga depicted patchy hyperfluorescent areas from the early phase and hypofluorescent dots in the late phase (fig . This patient was diagnosed with vkh disease and started to receive high - dose steroid pulse treatment . Methylprednisolone (1,000 mg / day) was intravenously administered for 3 days, followed by oral prednisolone for 16 weeks tapering off from 30 mg / day . Soon after the initiation of steroid treatment, the patient recognized a clearer visual field . The choroid became thinner (238 m in the right eye and 212 m in the left eye) and en face oct showed the clearer visualization of choroidal vessels bilaterally . However, 4 weeks after the cessation of steroids, bilateral disc swelling, thickened choroid (490 m in the right eye and 464 m in the left eye) and srf in the right eye recurred . Systemic steroids were started again and 4 weeks later, bilateral disc swelling resolved completely with the reduction of the choroidal thickness and srf . Two weeks later, however, srf in the right eye recurred under the oral steroid medication without disc swelling . No leakage was apparent on fa and no hypofluorescent dots were seen on icga . The choroidal thickness was unchanged with normal appearance on en face images; however, after sub - tenon's capsule triamcinolone acetonide (stta) injection (20 mg), srf resolved . Four weeks later, srf recurred again and to avoid the excessive use of steroids, bevacizumab was injected intravitreally; however, there were no remarkable changes . The additional stta and the oral steroids were effective and complete resolution of srf was obtained with improved vision of 1.0 . Four weeks later, however, srf recurred with pigment epithelial detachment (ped; fig . 2a) with a decreased bcva of 0.4 despite the continuation of the oral steroids and the stta, which were ineffective to resolve it . At this time, the choroid was not thick and en face oct depicted the choroidal vessels clearly (fig . Fa showed several leakages near the fovea and icga also depicted the leakage at the corresponding regions on fa with surrounding mixed hyper and hypofluorescence (fig . 2c, then, the eye was diagnosed with prednisolone - induced csc, and the intake of oral steroids was tapered . One month after the stopping of oral steroids, bilateral inflammation in the anterior chamber occurred with bilateral disc swelling . Oct showed thickened choroid (412 m in the right eye and 396 m in the left eye) with blurred choroidal vessels bilaterally, and then steroid therapy was restarted . Over 6 months under the low - dose oral steroids, this patient was initially diagnosed with vkh because of the bilateral disc swelling and characteristic hyporeflective dots on icga with thick choroid on oct . His symptoms were quite responsive to the systemic steroids . During the course, the srf in his right eye recurred several times without disc swelling, and it had been treated with steroids including stta, but gradually became refractory . Finally, the discontinuation of steroids was effective to resolve the fluid . From the fa and icga, the origin of the srf was considered to be choroidal vascular hyperpermeability such as csc which developed secondary to the steroids ., longer administration of steroids or stta may cause the development or worsening of csc . If steroids are continued even after the development of csc, they may worsen csc . Irreversible visual deterioration due to the damage of the rpe has been reported in the eyes with csc secondary to steroids . Therefore, we should know the early signs of the csc in eyes with vkh treated with steroids . So far, only two cases have been reported from japan in whom secondary csc occurred during the treatment for vkh . In each case, srf had relapsed during steroid tapering (prednisolone 15 and 5 mg, respectively), and diagnosed with csc using fa by the findings of dye leakages from the punctate hyperfluorescent spots seen at early phase . With the tapering of the steroids, srf promptly disappeared . Both reports concluded that angiography was the definitive tool to diagnose secondary csc . In vkh, the characteristic fa leakage is multifocal in the early stage followed by pooling of dye in the subretinal space . Otherwise in csc, univocal leakage from the level of rpe in the early stage is typical, but in eyes with csc associated with steroids, the intense leakage from multiple regions is often seen; therefore, in addition to fa, the evaluation of multimodal imaging including icga is essential to differentiate both diseases . On conventional oct b scans, retinal detachment is easily detected, but it occurs in both vkh and csc and sometimes it is difficult to distinguish them . The following are the differential points on oct: vkh accompanies the folds of rpe, fluctuation of inner limiting membrane and subretinal septa, while ped is more common in csc . The choroid is significantly thicker in vkh than that in csc . When diagnosing vkh and csc based on choroidal thickness, attention is required because the choroid is usually thicker in active vkh than in csc, but choroidal thickness changes during the course of vkh and at the cicatricial stage of vkh, the choroid becomes thin . In our case, the noninvasive en face oct scans of the choroid could suggest the different condition from active vkh as the cause of the recurrent srf . Every time showing the active vkh, the choroid was bilaterally thick and the choroidal vessels were blurred on en face oct images . On the contrary, when srf recurred without disc swelling, the choroidal thickness did not significantly increase and vessels were clearly traced, and such condition was considered to be associated with secondary csc . Through the course of this patient, en face choroidal imaging is helpful to understand the disease condition . Since the en face image is the same two - dimensional image as a conventional fundus photograph and angiography, it is easy to compare them . As histologically reported, the choroidal thickening in vkh is caused by the diffuse infiltration of lymphocytes, macrophages and epithelioid cells . Macrophages and polymorphonuclear neutrophils infiltrate into the choroid accompanied by interstitial edema and vascular dilatation . Such cellular infiltration may cause the blurry choroid on en face images . In csc, the choroid becomes thick due to the hyperpermeable choroidal vessels with an enlarged diameter but not blurry . The en face oct images of the choroid reflect those differences well and increase the accuracy of the diagnosis . Careful follow - up is warranted in vkh patients since secondary csc may develop during steroid treatment . En face oct observation of the choroid written informed consent to publish was obtained from the patient for publication of this case report and any accompanying images . A copy of the written consent is available for review by the editor of this journal.
Chronic liver diseases (cld) and its end - stages, cirrhosis and hepatocellular carcinoma (hcc), are leading causes of morbidity and mortality worldwide with enormous socio - economic costs . Patients with liver cirrhosis are at high risk of deadly hepatic failure and well over 80% of hcc develop on a cirrhotic background . Hcc ranks as the fifth most common cancer and, with over 600,000 deaths per annum, it constitutes a major global health problem (parkin et al . The main aetiologies of clds are chronic hepatitis c virus (hcv) and hepatitis b virus (hbv) infections, alcohol abuse and, as a result of metabolic syndrome reaching epidemic proportions, an increasing prevalence of non - alcoholic steatohepatitis (nash). Liver transplantation is currently the only available therapy for terminal liver failure . With donor organs being limited, preventive measures and prevention aims at eliminating the source of damage . In alcoholic liver disease, this obviously means avoiding further alcohol consumption . The addictive behaviour of patients, however, puts a serious limitation to the efficiency of this prevention strategy . Hbv and hcv infections, representing about one third of cld aetiologies, are combatted with virostatic treatments, thus improving patient conditions to some extent . The infections can, however, not be fully abrogated, severely reducing the efficiency of such therapy for this subset of clds . This liver injury triggers a cascade of molecular and cellular reactions geared towards damage limitation, removal or repair of damaged cells, defence against further infection, tissue repair and regeneration . Central to the natural response to injury is inflammation induced by a large battery of signalling molecules and executed by a variety of dedicated cells, repair by activated myofibroblasts, which produce the fibrous tissue and parenchymal cell proliferation, both of which are required for filling the holes caused by damage . Together, these cellular and molecular events release the enormous natural regenerative potential of the liver . In cld, hepatocyte damage, wound healing and tissue remodelling go awry, resulting in fibrosis and ultimately cirrhosis, the platform on which hcc and deadly hepatic failure develop . At the cellular and molecular level, the multistep process of progressive cld is reflected in the complex modulation of intracellular signal transduction circuits, altered cell - cell communications and, more drastically, an altered differentiation state of most liver resident cell types . Evidently, the dissection of these pathways is critical for the development of drugs and therapies . As is well recognised, the multifunctional cytokine transforming growth factor (tgf)- plays a pivotal role in the sequence of events leading to end - stage cld, although the complexity of the underlying aberrant responses that, in the various liver cell types and at the organ level, lead to the drastic changes seen in cld and hcc is not understood in detail (dooley et al . 2009; gianelli et al . 2011; matsuzaki 2009). To set the stage, we will first discuss the current knowledge of cellular communication and molecular mechanisms associated with cld initiation and progression and thereafter address the role of tgf- therein . The initial event is liver epithelial cell stress, resulting in necrotic and/or apoptotic death . Death - mediated signals induce the activation of kupffer and stellate cells, which orchestrate an inflammatory and wound - healing response that might lead to tissue regeneration and repair in an acute setting or to fibrogenesis, cirrhosis and cancer when injury occurs chronically . Below, we discuss the diverse phases that trigger disease progression and the process of liver regeneration . Cell damage and death clds are characterised by persistent hepatocyte damage and death, induced by either chemical toxicity, metabolic overload resulting in high levels of reactive oxygen species (ros) or viral / microbial activity causing metabolic deregulation (rombouts and marra 2010). In non - alcoholic and alcoholic fatty liver disease, this process is known as steatosis, which is (highly) reversible and does not necessarily lead to cell damage . In the case of cholestasis - mediated injury, bile duct epithelial cells (bdecs) rather than hepatocytes are the primary target of damage, bile reflux being the major inducer of this type of injury . Several modes of cell death have been classified in the damaged liver, including apoptosis and necrosis . Hepatocyte death triggers a cascade of reactions initiated actively by specific messengers and signalling molecules or simply by molecules released because of cell damage . The response is aimed towards damage limitation, removal or repair of damaged cells, wound closure, defence against further infection and tissue repair and regeneration . A highly interesting recent observation suggests that, upon liver damage, hedgehog ligand production is upregulated in dying liver epithelial cells to re - initiate a signalling pathway that controls progenitor cell fate and tissue construction during development, thus inducing progenitor cell expansion and liver regeneration (omenetti et al . 2011). Liver regeneration the liver possesses an enormous regenerative capacity . In experimental partial hepatectomy (phx) models, full liver mass is restored within 56 days after two - thirds phx in rats or mice . Key events and signalling pathways that control hepatocyte proliferation have been extensively investigated and have been summarised (michalopoulos 2007; bohm et al . 2010). However, in some settings, the potency of the toxic xenobiotic or the amount of damaged cells might cause the death of a large number of hepatocytes and cholangiocytes . Then, hepatic progenitor cells (hpcs) with bi - potential capacity, residing in the ductules and canals of hering, differentiate into either hepatocytes or cholangiocytes, depending on the cell compartment that is damaged the most (michalopoulos 2011). Rapid hepatic failure occurs if hepatic regeneration based on mature hepatocytes / cholangiocytes and hpc proliferation cannot replenish dead liver cells and liver architecture (duncan et al . Inflammation hepatitis is a central mechanism of disease progression and a marker of serious liver disease (weber et al . It encompasses a complex inflammatory response induced by a battery of signalling molecules and is executed by a variety of cells . The process is initiated by mutual activation of stellate and kupffer cells that together provide a cytokine milieu triggering massive infiltration of mononuclear cells, which include macrophages, lymphocytes, eosinophils and plasma cells . Lymphocytes are mobilised and stimulated by contact with antigen to produce lymphokines that further activate macrophages . Cytokines and chemokines from activated macrophages, in turn, stimulate lymphocytes, thus setting the stage for a persistent inflammatory response (wynn 2004; heymann et al . 2009).evidence is accumulating for the plasticity of liver macrophages, including kupffer cells, depending on the surrounding cytokine milieu: nf-b- and ap1-driven pro - inflammatory (m1) or stat6 and peroxisome proliferation - activated receptor - directed anti - inflammatory (m2) phenotypes are generated and correspondingly impact on subsequent cellular processes (vats et al . Fibrosis the activated (myo)fibroblast is the cell type responsible for wound closure and fibrosis (reflecting persistent wound - healing activity attributable to chronic damage) in any cld . Several potential sources for this critical mediator exist, including bone - marrow - derived fibrocytes or circulating mesenchymal cells, which can migrate through the injured liver and become myofibroblasts (friedman 2008; bataller and brenner 2005). Resident cells, e.g. Tissue fibroblasts located in the portal tract of the liver or quiescent hepatic stellate cells (hsc) located in the space of disse, might also be activated to become myofibroblasts (friedman 2000). Whether hepatocytes, cholangiocytes or even endothelial cells undergo a transition into activated fibroblasts under certain circumstances remains controversial (popov and schuppan 2010). The predominance of evidence still supports a central role for the quiescent hsc becoming activated by cytokine signalling, turning into myofibroblasts and then producing the fibrous scar that can be found in cld.cirrhosis is not simply extensive fibrosis but is characterised by architectural disruption, aberrant hepatocyte regeneration, nodule formation and vascular changes . The chance of a reversal from a cirrhotic to normal liver architecture remains controversial and corresponding data are discussed, for example by iredale (2007). On the other hand, no doubt exists that the resolution of liver fibrosis can occur, e.g. It is initiated by apoptosis and senescence as orchestrated biological processes to eliminate fibrogenic cells (friedman 2010; 2008).based on a multitude of data from rat or mouse hscs and animal models of liver damage, several conclusive statements about liver fibrosis can be drawn: (1) oxidative stress induces hepatocyte damage and hsc and kupffer cell activation, resulting in liver fibrosis, (2) tgf- is required for liver fibrosis and (3) the blunting of tgf- signalling reduces fibrogenesis . Carcinogenesis in cirrhotic livers, macroregenerative nodules that display foci of hepatocyte dysplasia are considered to be pre - neoplastic lesions of hcc . Histologically, these dysplastic lesions are classified as small cell or large cell lesions or as foci of adenomatous hyperplasia, whereby small cell dysplasia and adenomatous hyperplasia are the predominant preneoplastic lesions (roskams and kojiro 2010). Our present lack of a unified comprehensive understanding of liver carcinogenesis is partially attributable to hcc being initiated in multiple genetic and environmental contexts and almost certainly emerging as a consequence of multiple pathways (whittaker et al . This limitation regarding the pathogenesis of hcc has also prevented the development of effective, targeted, preventive or therapeutic interventions.recent analyses by using genome - wide approaches and improved animal models have initiated new and promising attempts at subclassifying the apparently highly heterogeneous hcc into distinct molecular and prognostic subtypes (lee and thorgeirsson 2006). The generally accepted paradigm of hepatocarcinogenesis is that malignant transformation occurs through the stepwise accumulation of genetic and epigenetic alterations that lead to the progressive acquisition of cancer phenotypes . On the other hand, more recent studies have led to the concept that a minimum number of molecular alterations leads to the acquisition of the key cancer phenotype, namely unconstrained cell proliferation (hoshida et al . Concept proposes that, in a physiological context, growth - promoting pathways are coupled with the activation of control mechanisms such as cellular senescence or apoptosis that limit their growth effects, producing a natural homeostasis of tissue mass . Additionally, the existence of cancer stem cells (csc) is now being intensely investigated in liver and hcc, since proliferation of stem cells is a frequent and permanent process in this organ and since the accumulation of adverse molecular events, e.g. Mutations, bears a high risk of csc generation (mishra et al . Such cancer stem cells, depending on their fate at the time point of carcinogenic conversion, could account for the development of hcc or cholangiocarcinoma (ccc).thus, in cld upon initial chronic liver damage, various progression phases can be distinguished in which a complexity of cellular crosstalk occurs . Tgf- has a pivotal role in orchestrating and regulating the corresponding phenotypes of cld (fig . 1). Upon liver damage, tgf- production is initiated in non - parenchymal liver cells, e.g. Granulocytes, macrophages (especially kupffer cells) and hsc (nakatsukasa et al . 1990), whereas fully differentiated epithelial cells do not express tgf-. Interestingly, hepatocytes appear to absorb and store significant amounts of latent tgf- in the cytoplasm and this might be activated and available upon damage, thus contributing to the initiation of hsc activation and the wound - healing process (roth et al . 1998). Of note, hepatocytes under culture conditions lose polarity and activate survival signalling pathways, thus gaining a fibroblastoid phenotype and the facility of autonomous tgf- production (dooley et al . 2008).fig . 1pros and cons of transforming growth factor- (tgf-) signalling during the progression of chronic liver diseases . Upon liver damage caused by many different aetiologies, active tgf- ligands show up in the liver and induce downstream signalling in all cell types investigated . Tgf- is recognised as a major profibrogenic cytokine and, thus, tgf--directed therapies are being investigated for their capacity to interfere with fibrogenesis and combat disease progression . Although many of these approaches have shown promising results in animal disease models for more than a decade, there is currently still no effective treatment for human disease on the market . This scheme attempts to explain the difficulties one faces when dealing with chronic liver diseases in human patients . In animal models, severe damage from fibrosis and inflammation can be achieved within weeks, whereas the establishment of end - stage liver disease in humans usually takes several decades to establish . During that life span, the liver undergoes many different phases, as shown along the central time line . Strongly depending on the disease stage, tgf- and thus its targeting might have a good (+) or bad () outcome in the organ . Tgf- enhances damage to epithelial cells by inducing apoptosis and oxidative stress, triggers myofibroblast (mfb) activation and a wound - healing response, controls or inhibits liver regeneration by hepatocyte apoptosis and inhibition of proliferation, activates regulatory t cells (treg) and th17 differentiation to calm down inflammatory responses, causes fibrogenesis and liver scarring in chronic disease, inhibits the proliferation of premalignant cells, activates stroma fibroblasts in the neighbourhood of tumour cells, inhibits tumour - directed inflammatory responses, facilitates tumour angiogenesis and induces epithelial - mesenchymal transition (emt) of tumour cells . This multiplicity of outcomes in one organ during the different stages of one disease clearly reveals the difficulties that we have to face while directing therapeutic approaches towards tgf-. One must select the accurate therapeutic window, target the right cell type and interfere with the adverse downstream branches of the signalling pathway . To achieve this, a great deal of basic research is still required pros and cons of transforming growth factor- (tgf-) signalling during the progression of chronic liver diseases . Upon liver damage caused by many different aetiologies, active tgf- ligands show up in the liver and induce downstream signalling in all cell types investigated . Tgf- is recognised as a major profibrogenic cytokine and, thus, tgf--directed therapies are being investigated for their capacity to interfere with fibrogenesis and combat disease progression . Although many of these approaches have shown promising results in animal disease models for more than a decade, there is currently still no effective treatment for human disease on the market . This scheme attempts to explain the difficulties one faces when dealing with chronic liver diseases in human patients . In animal models, severe damage from fibrosis and inflammation can be achieved within weeks, whereas the establishment of end - stage liver disease in humans usually takes several decades to establish . During that life span, the liver undergoes many different phases, as shown along the central time line . Strongly depending on the disease stage, tgf- and thus its targeting might have a good (+) or bad () outcome in the organ . Tgf- enhances damage to epithelial cells by inducing apoptosis and oxidative stress, triggers myofibroblast (mfb) activation and a wound - healing response, controls or inhibits liver regeneration by hepatocyte apoptosis and inhibition of proliferation, activates regulatory t cells (treg) and th17 differentiation to calm down inflammatory responses, causes fibrogenesis and liver scarring in chronic disease, inhibits the proliferation of premalignant cells, activates stroma fibroblasts in the neighbourhood of tumour cells, inhibits tumour - directed inflammatory responses, facilitates tumour angiogenesis and induces epithelial - mesenchymal transition (emt) of tumour cells . This multiplicity of outcomes in one organ during the different stages of one disease clearly reveals the difficulties that we have to face while directing therapeutic approaches towards tgf-. One must select the accurate therapeutic window, target the right cell type and interfere with the adverse downstream branches of the signalling pathway . To achieve this, hscs are a primary target for active tgf- in cld . Results of experiments in which tgf- is misexpressed in liver by using adenovirus or transgenic tgf- mice have revealed an important contribution of tgf- to hsc activation and fibrogenesis (kanzler et al . 2003). Upon its activation and phenotypic transdifferentiation, the tgf- response in this cell type changes . Whereas platelet - derived growth - factor - induced proliferation of quiescent hsc is antagonised by tgf-, transdifferentiated myofibroblasts demonstrate a growth stimulatory effect in response to tgf- (dooley et al . 2000). 1 (nieto et al . 2001) and 2 (inagaki et al . 1995) type (i) pro - collagen, tissue inhibitor of metalloproteinase-1 and 2 (herbst et al . 1997) and plasminogen activator inhibitor (pai)-1 (knittel et al . 1996) have been identified as direct tgf- target genes in this cell type, whereas the transcriptional activation of myofibroblast markers -smooth muscle actin (sma) and connective tissue factor (ctgf) are induced in a tgf--independent manner . Instead, tgf- signalling is required for -sma organisation and stress - fibre formation (dooley et al . 2003; uemura et al . 2005).quiescent hsc respond to tgf- treatment by smad activation and display a functional negative feedback regulation via the induction of smad7 . In contrast, myofibroblasts are fully stimulated via autocrine tgf- signalling and display a strong intrinsic r - smad activation and, importantly, lack smad7 upregulation (stopa et al . Tahashi and co - workers (2002) have confirmed this finding with myofibroblasts isolated from chronically injured rat livers; they conclude that the lack of smad7 induction as observed in myofibroblasts in cld could be one reason for excessive tgf- effects during the progression of liver fibrosis.several studies have identified smad3 as being the main mediator of the fibrogenic response of hsc, especially with respect to the induction of collagen expression (schnabl et al . 2001; furukawa et al . 2003; inagaki et al . 2001a; 2001b; seyhan et al . P38/jun amino - terminal kinase (jnk)/map kinase (mapk)-mediated smad3 linker phosphorylation has been reported to be associated with hsc migration and disease progression (yoshida et al . In addition to affecting the activin receptor - like kinase-5 (alk5)-smad3 pathway, tgf- has been found to mediate its profibrogenic action via the activation of an alternative type i receptor pathway in hscs, i.e. The alk-1-induced smad1 pathway mediating id1 expression . Ectopic overexpression of id1 enhances hsc activation, whereas its depletion blunts this tgf--induced response (wiercinska et al . 2006). Furthermore, non - smad tgf- signalling via ras, raf-1, mitogen - activated protein kinase kinase and mapk p42 and p44 signalling has been reported, although the outcome of these pathways on hsc activation and fibrogenesis has not been extensively studied (reimann et al . Hepatocytes the first investigations of tgf- effects on hepatocytes were performed in cultured cells and indicated that tgf- counteracted the stimulatory action on dna synthesis by various growth factors such as hepatocyte growth factor (hgf), epidermal growth factor (egf) or insulin (hayashi and carr 1985; nakamura et al . This tgf--induced growth arrest in hepatocytes was at least in part mediated via the interaction of smad proteins with sp1 transcription factors, which induce the expression of the cyclin - dependent kinase (cdk) inhibitor p21 (moustakas and kardassis 1998). (1986) showed that tgf- mrna increases in a late stage of the regenerating liver after phx . They further reported that, in the regenerating as in the normal liver, tgf- mrna is present in non - parenchymal cells but not in hepatocytes and the authors therefore suggested that tgf- was a component of a paracrine regulatory loop controlling hepatocyte replication (fausto et al . Subsequently, this cytostatic effect could also be shown in cultured hepatocytes from hepatectomised rat livers (strain et al . 1987).in addition to being anti - mitogenic, tgf- (and activin) has been found to induce hepatocyte apoptosis (schwall et al . The adaptor protein daxx, which is associated with the fas receptor that mediates the activation of jnk and programmed cell death induced by fas, was suggested to provide the connection between tgf- receptors and the apoptotic machinery (perlman et al . Tgf--induced apoptosis was shown to be mediated via smad - mediated induction of death - associated protein (dap) kinase (jang et al . 2002). Furthermore, tgf- has been shown to induce hepatocyte apoptosis via enhanced expression of the pro - apoptotic protein bim (ramesh et al . 2008).tgf- has been found, as for many other epithelial cell types, to have a biphasic role in hcc . Initially, in the primary tumour, tgf- has tumour - suppressive effects . In agreement with this notion, the reduced availability of tgf- type ii receptor (trii) by the ectopic expression of soluble trii in hepatocytes (kanzler et al . 2001) or by using heterozygous mice with reduced trii expression (i m et al . 2001) results in enhanced susceptibility to hcc, confirming the tumour - suppressor function of the tgf- signalling pathway . Ectopic expression of mutated ha - ras but not c - myc, has been found to abrogate the cytostatic effect of tgf- on hepatocytes (houck et al . Hcc cells might become selectively resistant to the cytostatic effects of tgf- (braun et al . 1990) and not lose all responses to tgf-, such as epithelial - to - mesenchymal transition (emt). These hepatocytes display a high degree of differentiation and undergo cell cycle arrest in the g1 phase following exposure to tgf-. They maintain epithelial polarisation, despite the expression of oncogenic ha - ras; however, upon tgf- stimulation, they convert into a migratory cell type with fibroblastoid morphology and proliferation is no longer inhibited by tgf-. Without oncogenic ras, in a preneoplastic setting, tgf- might activate the epidermal growth factor receptor (egfr) and induce c - src phosphorylation leading to akt activation and cell survival . The blocking of egfr signalling then amplifies the apoptotic response to tgf-, whereas tgf--mediated emt in hepatocytes is unaffected (murillo et al . 2005). This indicates that the activation of egfr is required for impairing cytostatic tgf- activity but is dispensable for the emt process.using monolayer and sandwich culture systems, we have shown that hepatocytes can exist in differentiated and dedifferentiated states that are reversible and can be switched by manipulating the responsible key factors of the signalling network . For example, focal adhesion kinase - mediated akt and extracellular signal - regulated kinase 1/2 signalling interferes with the cytostatic effects of tgf-, thus facilitating fibroblastoid transdifferentiation (emt). Abrogating survival signalling resensitises hepatocytes to tgf--induced apoptosis (godoy et al . In addition, immortalised, highly differentiated hepatocytes, when treated with tgf-, maintain their epithelial morphology and undergo dramatic alterations in adhesion, leading to detachment, re - adhesion and spreading . These alterations in adhesive behaviour are caused by sequential changes in the expression of 51 integrin and its ligand fibronectin (biname et al . 2008). Ctgf is potently induced by tgf- and both factors are thought to play an important role and to cooperate in fibrogenesis and emt (wang et al . 2011; gressner and gressner 2008).the results described in the preceding paragraph and from others (kaimori et al . 2007; zeisberg et al . 2007) show that tgf-, e.g. Via the induction of the transcriptional repressor snail, induces adult mouse hepatocytes to undergo phenotypic and functional changes typical of emt . Whether, however, hepatocyte emt occurs in vivo during fibrogenesis is currently under discussion . (2007) have performed lineage - tracing experiments with albcre - r26rstoplacz double - transgenic mice and demonstrated that hepatocytes undergoing emt substantially contribute to the population of fibroblast - specific protein-1 (fsp1)-positive fibroblasts in carbon tetrachloride (ccl4)-induced liver fibrosis . Moreover, rowe et al . (2011) have demonstrated that hepatocytes up - regulate the expression of snail in vivo during tissue remodelling . Hepatocyte - specific ablation of snail demonstrates that this transcription factor plays a key role in liver fibrosis progression in vivo by triggering multiple aspects of fibrogenesis, including growth factor expression, extracellular matrix (ecm) synthesis and chronic inflammatory responses (rowe et al . (2010) have strongly challenged the concept that hepatocytes in vivo acquire a mesenchymal phenotype through emt to produce ecm in liver fibrosis . When triple - transgenic mice expressing rosa26 stop -galactosidase, albumin cre and collagen 1(i) green fluorescent protein (gfp) and in which hepatocyte - derived cells are permanently labelled by -galactosidase and type i collagen - expressing cells are labelled by gfp are subjected to the induction of liver fibrosis by repeated ccl4 injections, no cells with double - positivity for gfp and -galactosidase can be found, although all -galactosidase - positive cells exhibit abundant cytoplasm and the typical morphology of hepatocytes and express none of the mesenchymal markers including -sma, fsp1, desmin and vimentin . The authors conclude that type i collagen - producing cells do not originate from hepatocytes and that hepatocytes in vivo neither acquire mesenchymal marker expression nor exhibit a morphological change that is distinguishable from normal hepatocytes (taura et al . Wells and coworkers present even more convincing results that, in alfp - cre 3 rosa26-yellow fluorescent protein (yfp) mice, neither hepatocytes, nor cholangiocytes (or their bipotential progenitors) display evidence for in vivo colocalisation of yfp with mesenchymal markers s100a4, vimentin, -sma or procollagen 1 2 in three animal models of chronic liver disease (bile duct ligation, ccl4 and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (chu et al . 2011).reconciling these apparently contradictory findings will require further investigations . Nevertheless, if transgenic mice with upregulated smad7 expression only in their hepatocytes are challenged with chronic exposure to ccl4, they demonstrate significantly diminished liver damage and fibrosis when compared with controls (dooley et al . 2008), indicating that tgf- signalling in hepatocytes in vivo is required for fibrogenesis progression, as it is in hsc . Other cell types of the liver much less intensely investigated are the effects of tgf- on other cell types of the liver, e.g. Bdec, immune cells and liver sinusoidal endothelial cells, although a major influence can be expected in liver physiology and during cld . Similar to hepatocytes, tgf- might provide cytostatic and tumorigenic effects towards bdec, in particular during carcinogenic progression in cholangiocarcinoma . From many studies in other tissues, an inhibitory role of inflammation is expected, especially in its role as a differentiation factor for regulatory t cells (tregs; becker et al . Finally, the impact of tgf- as a pro - angiogenic factor has been well - described (ten dijke and arthur 2007) and will be relevant in the branched sinusoid network of the liver in the settings of regeneration, cirrhosis and carcinogenesis . Various strategies have been pursued to accomplish the inhibition of tgf- signalling in fibrosis (fig . 2), first in animal studies and then with the objective of being translated into humans (see next paragraph). These include (1) sequence - specific anti - sense oligonucleotides that inhibit tgf- mrna expression, (2) isoform - selective neutralising antibodies, soluble trii fragments or synthetic peptides that interfere with ligand binding to the endogenous receptor complex, (3) overexpression of the natural tgf- signalling inhibitor smad7 or cytokines such as interferon- (ifn-) that induce smad7 expression, (4) neutralising antibodies to integrins that interfere with the activation of latent tgf- and (5) low - molecular - weight inhibitors antagonising the intracellular kinase activity of tgf- receptors (iyer et al . 2005; pennison and pasche 2007; yingling et al . 2004; dooley et al . 2003; petersen et al . 2008; weng et al . 2007; hawinkels and ten dijke 2011). Such anti - tgf- approaches have been established and successfully used for the treatment of experimental fibrogenesis . For example, a synthetic peptide that blocks the interaction of tgf- with its receptor has been established to be effective in protection against ccl4-induced liver fibrosis (ezquerro et al . Further, dominant - negative or soluble triis have been applied to suppress fibrosis in mice and rats upon dimethylnitrosamine-, ccl4- or bile duct ligation - mediated liver damage (qi et al . 2002). In part, the inhibitory effect of soluble trii is achieved by interference with oxidative stress, including the generation of ros (cui et al . Similarly, tgf--binding proteins, such as decorin and antagonistic cytokines, such as bone morphogenetic protein-7, hepatocyte growth factor, interleukin-10 or ifn- were as efficient as camostat mesilate, a protease inhibitor that possibly abrogates the proteolytic activation of tgf- (breitkopf et al . 2005). Moreover, adenovirus - mediated overexpression of smad7 in the liver potently blunted bile duct ligation - induced liver fibrosis and achieved efficient inhibition of intracellular tgf- signalling . Bile duct ligation induced profibrogenic effects in cultured hscs and in vivo were inhibited (dooley et al . 2003). A similar approach with adenovirus - mediated overexpression of smad7 successfully interfered with bleomycin - induced lung fibrosis in mice (tgf- signals via heteromeric transmembrane complexes of type i and type ii receptors (tr) that are endowed with intrinsic serine / threonine kinase activity (alk activin receptor - like kinase). Upon type - ii - mediated phosphorylation of the type i receptor, the activated type i receptor initiates intracellular signalling by phosphorylating receptor regulated (r)-smad2 and smad3 . Activated r - smads form heteromeric complexes with smad4 and these complexes accumulate in the nucleus where they mediate transcriptional responses . Inhibitory smad7 antagonises tgf-/smad signalling by competing with r - smads for receptor interaction and by recruiting e3 ubiquitin ligases to the activated receptor complex and mediating its degradation . This pathway has been targeted by anti - sense molecules that inhibit tgf- mrna expression, by neutralising antibodies against tgf- or tgf- receptors that interfere with ligand - receptor interactions, by antibodies that interfere with the activation of latent tgf- and by soluble extracellular domains of the type ii receptor that sequester ligand binding to endogenous receptors and small atp mimetics of tgf- receptor kinases . Antagonising pathways, such as interferon- (ifn-), tumour necrosis factor- (tnf-) and epidermal growth factor (egf), can inhibit tgf-/smad - induced responses by stimulating smad7 expression tgf- signal transduction pathway and targets for therapeutic intervention . Tgf- signals via heteromeric transmembrane complexes of type i and type ii receptors (tr) that are endowed with intrinsic serine / threonine kinase activity (alk activin receptor - like kinase). Upon type - ii - mediated phosphorylation of the type i receptor, the activated type i receptor initiates intracellular signalling by phosphorylating receptor regulated (r)-smad2 and smad3 . Activated r - smads form heteromeric complexes with smad4 and these complexes accumulate in the nucleus where they mediate transcriptional responses . Inhibitory smad7 antagonises tgf-/smad signalling by competing with r - smads for receptor interaction and by recruiting e3 ubiquitin ligases to the activated receptor complex and mediating its degradation . This pathway has been targeted by anti - sense molecules that inhibit tgf- mrna expression, by neutralising antibodies against tgf- or tgf- receptors that interfere with ligand - receptor interactions, by antibodies that interfere with the activation of latent tgf- and by soluble extracellular domains of the type ii receptor that sequester ligand binding to endogenous receptors and small atp mimetics of tgf- receptor kinases . Antagonising pathways, such as interferon- (ifn-), tumour necrosis factor- (tnf-) and epidermal growth factor (egf), can inhibit tgf-/smad - induced responses by stimulating smad7 expression as mentioned above, experimental evidence exists showing that smad3 is the predominant mediator of fibrogenic tgf- downstream signalling . Therefore, strategies to interfere specifically with smad3 are promising . In line with this assumption, the targeted disruption of smad3 confers resistance to the development of dimethylnitrosamine - induced hepatic fibrosis in mice (latella et al . 2002). However, small molecule inhibitors announced as specific for smad3 (jinnin et al . 2006) have not yet yielded breakthrough results and seem to need further improvement . With regard to interference with cancer, recent studies in g. giannelli s lab (giannelli et al . 2011) have shown that the inhibition of tgf- signalling results in multiple synergistic downstream effects that probably improve the clinical outcome in hcc . Further, the small molecule inhibitor ly2109761, which targets tri / alk5 and trii induces a complete abrogation of smad - dependent and -independent signalling in human colon carcinoma cells harbouring activated k - ras, resulting in reduced tumour cell invasion and liver metastasis (zhang et al . Contrary to the stimulating beneficial outcome of anti - tgf- treatment in animal disease models (breitkopf et al . For example, metelimumab, a monoclonal antibody against tgf-1, has been used to treat systemic sclerosis . Other studies involving the use of lerdelimumab, a monoclonal antibody against tgf-2, to treat eye scarring (mead et al . 2003), or gc1008, a pan - antibody against tgf-1 - 3 for lung fibrosis are still ongoing . Intriguingly, an antisense strategy against tgf-2 has been successful in the treatment of glioma and is currently being tested in other malignant tumours such as pancreatic carcinoma, colon carcinoma and melanoma (schlingensiepen et al . A major reason that most of these promising results from animal disease models have not yet robustly been translated into clinical use is probably the multiplicity of the possible biological - context - dependent functions of tgf-. In the time course of cld progression, various phases occur, such as initiation, regeneration, perpetuation, fibrogenesis, tumorigenesis and metastasis . Depending on the specific disease stage,, it triggers the transdifferentiation of hsc to myofibroblasts and thus mediates a wound - healing response . During regeneration and hepatocyte proliferation, tgf- has an important tissue - mass - limiting cytostatic effect and controls inflammation by generating tregs . During perpetuation and fibrogenesis in chronic disease stages, the overwhelming scar - forming wound - healing reaction is adverse for the liver . In the pre - malignant stage,, the negative outcome of tgf- on inflammation might inhibit the immune response against arising tumour cells . In carcinogenesis, when tgf--cytostatic effects are lost and the signalling branch is redirected to emt, tgf- may favour cancer progression and metastasis . Furthermore, the pro - angiogenic action of tgf- towards endothelial cells may also be important for tumour progression (meyer et al . Thus, an important consideration for tgf--directed treatment of fibroproliferative diseases, such as cld, is to select the right time point and cell type for targeted intervention (fig . 1). Moreover, an additional challenge is that the selective inhibition of some but not all tgf--induced cellular responses might be beneficial.
Ewing sarcoma / primitive neuroectodermal tumor (es / pnet), previously thought to be separate tumors, is now treated as the same tumor; both have similar immunohistochemical characteristics and chromosomal translocation . They are malignant tumors composed of undifferentiated small round cells, usually affecting children, adolescents, and young adults . Generally es / pnet affects the bones and deep soft tissues, although other organs such as the pancreas, small bowel, esophagus, kidneys, prostate, ovaries, vagina and rectovaginal septum have been reported; this is termed as extraskeletal es / pnet . To the best of our knowledge, only 5 cases of gastric es / pnet have been reported in the english language literature . A 31-year - old healthy female patient was admitted to the surgical ward due to upper abdominal pain and coffee ground vomiting of 3 days duration . The patient had no other complaints and was hemodynamically stable . Rectal examination revealed melena . A nasogastric tube was inserted and revealed coffee ground secretions . Upper endoscopic examination revealed a large ulcerated mass located at the lesser curvature of the stomach, with oozing of blood . Biopsy revealed tumor cells showing positive immunoreactivity for cd99 (fig 1), fli1, vimentin, and ki67, and negative immunoreactivity for cytokeratin, s100, cd20, cd3, cd79a, pax5, cd30, cd43, dog-1, cd68, cd163, cd33, mpox, and desmin . Es / pnet was suspected and fluorescence in situ hybridization (fish) analysis was ordered, which was positive for the ewsr1 gene rearrangement (11: 22 translocation). Total body computed tomography (ct) showed a hypodense mass measuring 9 cm at the lesser curvature of the stomach, with compression on the splenic vein (fig 2). Positron emission tomography - ct (pet - ct) revealed pathological uptake of fluorodeoxyglucose at the gastric mass and lymph nodes at the gastrohepatic ligament (fig 3). The patient refused neoadjuvant treatment, and thus surgery was performed . On exploration of the abdomen, the mass was adhering to the pancreatic tail and mesentery of the transverse and descending colon, along with abnormal pathological lymph nodes at the greater curvature . Histopathological examination revealed the mass measuring 11 cm in diameter to be an es / pnet invading the gastric wall, pancreas, and splenic hilum, without involvement of the left adrenal . Three years postoperatively, the patient is doing well, with no evidence of disease recurrence . Es, a term used to describe tumors that lack neuroectodermal differentiation, and pnet, used to describe tumors that exhibit neuroectodermal features, are now treated as a single entity . Tumor cells are rich in glycogen, and pseudorosette formation characterizes the tumor's morphological differentiation . The diagnosis can be made by immunohistochemical staining for a monoclonal antibody to cd99 (hba/71, 12e7, and 013), which is positive in almost all cases of es / pnet [5, 6, 7]. Another immunohistological reagent that can be used for diagnosis is the intermediate filament vimentin, which is usually positive . Markers showing variable immunohistochemical staining include s100, chromogranin a, synaptophysin, and neuron - specific enolase . For tumors occurring in older patients or at unusual sites, fish or reverse transcription polymerase chain reaction can be used for diagnosis [3, 8]. These are used to test for the presence of genetic mutation (11: 22)(q24:q12) translocation (ews / fli1 fusion), which is an essential criterion for the diagnosis of es / pnet, although sometimes these tests are negative [9, 10]. Due to the poor results of surgery alone as a treatment modality for extraskeletal es / pnet, the current recommendation is multimodal treatment including surgery, chemotherapy, and radiotherapy . The chemotherapeutical agents used as standard therapy for es / pnet include vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide . Of these 5 cases, 3 were females and 2 males, with an average age of 46.6 years (range 1468 years) and an average tumor size of 8.5 cm . Most of these patients presented with abdominal pain, and 1 presented with an abdominal mass . Two cases had hepatic metastasis, 1 had lymph node and peritoneal metastasis, 1 no metastasis, and 1 was not reported . Only 2 patients (of the 3 with metastasis) herein, we describe the 6th case of es / pnet of the stomach . Due to the patient's refusal of chemotherapeutical treatment three years postoperatively, the patient is doing well, with no evidence of disease recurrence . Primary gastric es / pnet is a very rare tumor, and few cases are reported in the literature . Due to its rarity, written informed consent was obtained from the patient for publication of this case report and accompanying images; it is available for consultation . They confirm that the manuscript has not been published elsewhere and is not under consideration by another journal.
T1 dm is an autoimmune - mediated disease characterized by selective destruction of insulin - producing pancreatic -cells, resulting in the need for lifelong administration of exogenous insulin for patient survival, and represents 510% of all cases of diabetes [2, 3]. The pathological condition begins with an autoimmune inflammatory process known as insulitis, which leads to leucocyte infiltration into the pancreatic islets of langerhans, resulting in the autoimmune destruction of pancreatic -cells . Thus, the onset of t1 dm is the result of the recognition of self - antigens due to molecular mimicry against infection with various viruses such as coxsackie virus, -cell cytotoxicity, or -cell cytolysis [57]. Consequently, molecules such as preinsulin, insulin, glutamate decarboxylase-65 (gad-65), and islet antigen 2 become recognized as self - antigens . Different types of innate immune cells such as t cells, eosinophils, macrophages (m), and dendritic cells (dc) as well as proinflammatory cytokines and chemokines are present during insulitis [4, 9]. The relationship between t1 dm and high levels of inflammatory cytokines such as interleukin- (il-) 1 [1014], interferon- (ifn-), tumor necrosis factor- (tnf-), il-12 [17, 18], and macrophage migration inhibitory factor (mif) [1924] has been widely recognized . Mif is a pleiotropic cytokine produced during the immune response by activated t cells, m, dc, and a variety of nonimmune cells and plays a pivotal role in the systemic inflammatory immune response by promoting the production of proinflammatory cytokines including tnf- and il-6, which are involved in inflammatory and autoimmune diseases such as septic shock, cancer, inflammatory bowel disease [27, 28], rheumatoid arthritis [29, 30], obesity [31, 32], and diabetes [3335]. Moreover, mif has recently been proposed as a diagnostic biomarker for autoimmune diseases such as arthritis [37, 38], type 2 diabetes, and ulcerative colitis in both animals and humans . The pathogenic contribution of mif to t1 dm was demonstrated by showing that mif mrna expression was upregulated in splenic lymphocytes during the development of spontaneously diabetic nonobese diabetic (nod) mice, as well as cyclophosphamide - treated nod mice . Diabetes incidence was increased to 86% in nod mice treated with recombinant mif (rmif) protein, compared with the 55% incidence observed in untreated control nod mice . Furthermore, studies performed using mif/ mice rendered diabetic by administering multiple low doses of streptozotocin (stz) have shown that the absence of mif affected several aspects of experimental t1 dm, including initial immunopathological events and the production of proinflammatory and cytotoxic mediators, thereby interfering with both inflammation and tissue destruction . All the results described above provide evidence that mif plays a critical role in the pathogenesis of t1 dm . However, the precise mechanism by which mif promotes insulitis and the mechanism underlying its proinflammatory role remain unclear . The activities of mif may reside at the levels of both the inductive and effector phases of the inflammatory response attributed to antigen - presenting cells . Here, we analyzed the influence of mif on m and dc activation using an autoimmune diabetes model in which multiple low doses of stz were administered to mif/ and wild - type (wt) mice (mif/stz and wtstz, resp .) In the balb / c genetic background . As previously demonstrated mif/stz developed less severe hyperglycemia, reduced levels of ifn- and tnf-, a smaller amount of pancreatic islet antigen- (piag-) specific igg, and decreased cell infiltration into the pancreatic islets compared to wtstz . Interestingly, we found for the first time that m and dc from mif/stz displayed decreased expression of costimulatory molecules cd80, cd86, and cd40, as well as toll - like receptor- (tlr-) 2, tlr-4, and major histocompatibility complex- (mhc-) ii . Importantly, we demonstrated that due to diminished upregulation of costimulatory molecules, these cells exhibited a reduced capacity to induce proliferation and cytokine expression in cocultures with allogeneic ovalbumin- (ova-) specific t cells . All experiments in this study were performed according to the guidelines in the mexican regulations on animal care (nom-062-zoo-1999, 2001) and were approved by the local institutional animal care and use committee . Six- to 8-week - old male balb / c mice were purchased from harlan laboratories (mexico city, mex .) And were maintained as a breeding colony in a pathogen - free environment at our animal facilities in accordance with institutional guidelines . Abhay r. satoskar (the ohio state university, usa) and were maintained as breeding colonies for more than 10 generations in the balb / c genetic background on the transgenic mouse core facility at our institution . Genotyping of mif/ mice was routinely performed on dna isolated from tail snips using a pcr procedure . The pcr were performed using the following primers: mif: forward 5-agaccacgtgcttagctgag-3 and reverse 5-gcatcgctaccggtggataa-3; neomycin (neo): forward 5-attgaacaagatggattgcac-3 and reverse 5-cgtccagatcatcctgatc-3. Pcr for the amplification of mif and neo was performed by adding 100 ng of the extracted dna to 25 l of a reaction mixture that contained 18.4 l of distilled water, 2.5 l of 10x pcr buffer, 0.4 l of dntps (10 mm), 1.5 l of mgcl2 (25 mm), 1 l of the forward and reverse primers, and 0.2 l (2.5 units) of taq dna polymerase (ampliqon, bioreagents and molecular diagnostics). The amplification protocol consisted of 5 min at 95c, 35 cycles of 95c for 30 sec, 58c for 40 sec, and 72c for 30 sec and a final extension at 72c for 5 min . A pcr fragment of 200 bp, corresponding to mif, or 500 bp, corresponding to neo, was visualized to identify wt or mif/ mice, respectively . The pcr products were analyzed by electrophoresis on a 1.5% agarose gel and were viewed under uv light (bio - rad, usa). Mif/ and wt mice were deprived of food for 8 h before induction of diabetes via intraperitoneal (i.p .) Injection of stz at doses of 40 mg / kg of body weight, daily for five consecutive days (days 04) (sigma - aldrich, st . Stz was diluted in cold 0.01 m citrate buffer (ph 4.5) and was used within 5 min of preparation, in accordance with a previously reported protocol . Blood samples were collected by tail snipping from wt and mif/ mice that had been fasting for 6 h. samples were obtained once before stz injection and 2, 4, and 8 weeks after stz injection . Blood glucose levels were measured with a portable glucometer (accu - chek sensor glucometer; roche diagnostics, indianapolis, in, usa). Mice with a glucose concentration exceeding 300 mg / dl were considered to have t1 dm . Blood was collected and centrifuged at 1300 g, and the serum levels of mif (neobiolab, usa), il-12, ifn-, il-17, il-4, il-13 (peprotech, mex), and insulin (lincoln, st . Charles, mo, usa) were determined via elisa according to the manufacturer's instructions . After isolation, the islets were lysed by five freeze - thaw cycles followed by sonication (six 60-hz cycles for 1 min each) on ice . After centrifugation (10 000 g, 15 min, 4c), supernatants were collected and filtered through a 0.2 m membrane (corning, cambridge, ma, usa). The protein concentration was determined using the lowry method, and piag aliquots were stored at 70c until use . Serum samples were analyzed for the levels of pancreatic islet - specific th1-associated igg2a and th2-associated igg1 antibodies by elisa . Briefly, 96-well elisa plates (costar) were coated with 100 l / well soluble piag in tris buffer, ph 7.8 ., the wells were washed thoroughly with phosphate - buffered saline (pbs) containing 0.05% tween-20 (pbs - tween; merck, france) and were blocked with pbs supplemented with 1% bovine serum albumin (pbs - bsa; sigma - aldrich) for one hour at room temperature (rt). The serum samples were diluted 1: 100, followed by serial dilution of each sample (in pbs - bsa) from healthy or stz - treated wt or mif/ mice . After extensive washing with pbs - tween, the samples were incubated for 45 min at rt with isotype - specific peroxidase - labeled goat anti - mouse antibodies (anti - igg1 and anti - igg2a at 1/1000 dilutions; zymed, san francisco, ca, usa). Then, the plates were washed, and immunoreactivity was detected with abts solution (zymed). Pancreatic tissues from wt and mif/ mice healthy or injected with stz were removed, fixed overnight in 4% formaldehyde, and embedded in paraffin blocks . Afterwards, 5 to 7 m transverse sections of pancreatic tissue were sliced from the paraffinized tissue blocks, mounted on slides, and subsequently stained with eosin - hematoxylin (e&h; sigma - aldrich). For each mouse, one histological containing 13 nonsuccessive slices was scored for infiltration as previously described according to the following scale: grade 0 (no insulitis) = 0% infiltration within the islets; grade 1 (peri - insulitis) = 110% infiltration; grade 2 (moderate insulitis) = 11<50% infiltration; grade 3 (severe insulitis) => 50% infiltration; or grade 4 (complete insulitis) = extensive infiltration with few or no detectable pancreatic islet cells . Using an olympus bx51 microscope (olympus america, melville, ny, usa) equipped with a digital video camera, spleen and pancreatic islet cells from wtstz and mif/stz were collected after 0, 2, 4, and 8 weeks of injection with stz and stained for flow cytometry analysis . Briefly, the spleen was removed under sterile conditions, and spleen cells were obtained by mincing and filtering the tissue, followed by washing and suspension in dmem culture medium supplemented with 10% fetal bovine serum, 100 units of penicillin / streptomycin, 2 mm glutamine, and 1% nonessential amino acids (all from gibco, brl, grand island, ny, usa). Spleen cells were suspended at 5 10 cells / ml in the same medium . The pancreas was also removed under sterile conditions, and pancreatic islets were isolated using the collagenase method as previously described . Briefly, the pancreas was removed and cut into small pieces (approximately 3 mm in size). The tissue was subsequently incubated in collagenase (0.3 mg / ml; roche diagnostics corp ., indianapolis, in, usa) for 10 minutes at 37c in a total of 1 ml of digestion solution under constant shaking and intermittent vortexing . Islets were subsequently washed several times in hbss containing bsa (5 mg / ml) and were hand - picked under a dissecting microscope . Islets were dispersed into single cells by suspension in trypsin - edta (gibco, brl) and passage through a siliconized pasteur pipette . Cells from the spleen or pancreas obtained as described above were then used for flow cytometry analysis . In brief, cells were washed in flow cytometry wash solution (dulbecco's pbs containing 1% fcs and 0.05% sodium azide), followed by incubation with allophycocyanin- (apc-) conjugated anti - f4/80 and anti - cd11c antibodies for differentiation of m and dc, respectively . Then, the selected cells were incubated in 3% bsa - pbs containing a phycoerythrin- (pe-) labeled anti - cd80, anti - ccr5, or anti - tlr-4 antibody or a fluorescein isothiocyanate- (fitc-) labeled anti - cd86, anti - cd40, anti - mhc - ii, or anti - tlr-2 antibody (all antibodies from biolegend, san diego, ca, usa) at 4c for 30 min . After incubation, the cells were washed several times in buffer, fixed in 1% paraformaldehyde (sigma - aldrich), and stored at 4c in the dark, followed by analysis using a facscalibur flow cytometer and cellquest software (becton dickinson, franklin lakes, nj, usa). Adherent m among peritoneal exudate cells (pecs) from healthy or 8 weeks after stz - treatment wt or mif/ mice were obtained . Briefly, the m density was adjusted to 5 10 cells / ml, and the cells were plated (100 l) in 96-well flat - bottom plates (costar, cambridge, ma, usa). Three hours later, the pecs were washed three times with warm sterile pbs to remove nonadherent cells, and 10 g of ova (worthington, usa) in 100 l of dmem supplemented media was added . Three hours later, adherent m were washed three times with warm sterile dmem to remove excess ova that had not been phagocytosed . Spleen cells from ova - transgenic mice were obtained as previously described, suspended at 1 10 cells / ml, and added (100 l) to pecs at a ratio of 1 m: 5 spleen cells . Then, [3h]-thymidine (185 gbb / mmol activity, amersham, uk) was added at 0.5 ci / well, and the cells were incubated for a further 18 h. the cells were harvested using a 96-well harvester (tomtec, toku, finland) and then counted using a microplate counter (trilux, toku, finland). The values are presented as counts per minute (cpm) from triplicate wells . To establish the mif levels under conditions of t1 dm, the serum mif levels were determined weekly until 8 weeks after stz treatment in wtstz . Mif/stz received similar concentrations of rmif (r&d systems, usa) to the mif levels observed in wtstz to emulate physiological conditions in wtstz . Injection of stz together with 500 pg of rmif in 100 l of saline solution as a vehicle . Injection of rmif every three days at the following doses: 850 pg on week 1; 1280 pg on week 2; 1292 pg on week 3; 1305 pg on week 4; 4300 pg on week 5; 7300 pg on week 6; and, finally, 13200 pg on week 7 . Other mif/stz mice were injected with an equivalent volume of saline solution (100 l) as controls . Comparisons between wt and mif/ mice that were healthy or treated with stz were performed using either student's unpaired t - test or anova followed by turkey's multiple comparisons test for data that displayed a normal distribution . P values less than 0.05 were considered significant and were designated as p <0.05, p <0.01, or p <0.001 . All data were analyzed using graphpad prism 6 software (san diego, ca, usa). These mice sustained high blood glucose levels from week 2 (281.7 16 mg / dl) until week 8 of stz administration (470.5 24 mg / dl) (figure 1(a), wtstz: squares). In contrast, hyperglycemia developed gradually in mif/stz . The blood glucose level of mif/stz peaked at 219 23 mg / dl on week 4, and the blood glucose level in some mice decreased between weeks 6 and 8 (226 15 and 182 16 mg / dl, resp .) (figure 1(a), mif/stz: inverted triangles). This observation suggested that a slight recovery of the glucose response may occur in mif/stz at approximately week 8 of stz treatment . Wtstz showed a peak blood insulin level at 2 weeks after stz treatment, and after the sixth and eighth weeks of treatment, their blood insulin levels were significantly lower than those of healthy wt mice (figure 1(b), wtstz: squares with solid line; wt mice: white circles with dotted line). Interestingly, mif/stz showed no significant changes in insulin levels compared to healthy mif/ or wt mice over the course of the experiment (figure 1(b), mif/stz: inverted triangles with solid line; healthy mif/ mice: white triangles with dotted line). We measured the serum levels of proinflammatory and anti - inflammatory cytokines in wt and mif/ mice after stz administration . As expected, the wtstz displayed gradually increasing serum levels of inflammatory cytokines such as mif, il-12, and ifn- (figures 2(a), 2(b), and 2(c), resp . The level of the proinflammatory cytokine il-17 was significantly increased at 8 weeks after t1 dm induction in wtstz (figure 2(d), squares). In contrast, mif/stz displayed significantly lower serum levels of mif, il-12, ifn-, and il-17 than wtstz (figures 2(a), 2(b), 2(c), and 2(d), resp . ; the serum levels of the anti - inflammatory cytokine il-4 were significantly lower in wtstz (squares) than in mif/stz (inverted triangles) (figure 2(e)). No differences in the serum il-13 levels were found between wtstz and mif/stz (figure 2(f)). To confirm that stz reached its target, wt and mif/ mice were treated with a single high dose of stz (150 mg / kg). The toxic effect of high - dose stz was similar between wt and mif/ mice (supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/7053963). In both experimental groups, the toxic effect of stz was present, and the high dose of stz destroys the insulin - producers beta cells, leading to acute nonimmune - mediated diabetes; in contrast the multiple low doses of stz at 40 mg / kg, daily for five consecutive days, require participation of immune - inflammatory events for t1 dm development . To assess the damage to cells in a model of t1 dm induced by multiple low doses of stz, we sacrificed the mice at 8 weeks after t1 dm induction and removed the pancreas for h&e staining and histological analysis . We assessed the number and size of normal and infiltrated pancreatic islets in each slide for the experimental and healthy mice . Islets from healthy wt and mif/ mice had a round morphology and well - defined borders without cellular infiltration (figures 3(a) and 3(c)). As expected, the pancreas from wtstz showed fewer and smaller islets than those from healthy wt mice . Additionally, evident cellular infiltration led to the breakdown of islet morphology in the pancreas of wtstz (figure 3(b)). In contrast, partial islet damage (figure 3(d)) and no significant reduction in islet number or size were observed in mif/stz compared to wtstz (table 1). As shown in figure 3(e), mif/ mice at 8 weeks after stz administration displayed marked reductions in both invasive insulitis (insulitis grades 3 and 4) and mild peri - insulitis (insulitis grade 2) in pancreatic islets compared to wtstz . Thus, pancreatic islets from mif/stz had reduced damage associated with reduced leucocyte infiltration compared to pancreatic islets from wtstz . These results demonstrated that stz administration triggers -cell destruction followed by insulin deficiency and hyperglycemia in wt mice . Although stz reached the pancreatic islets in mif/ mice, mif/stz exhibited minor damage compared to wtstz . In t1 dm, the immune system targets self - antigens within pancreatic islets and destroys the inhabiting insulin - secreting -cells . Therefore, autoantibody detection serves as a predictive factor for the onset of diabetes in both humans and mice [46, 47]. To investigate how mif contributes to autoantibody production in t1 dm, the serum levels of pancreatic islet - specific igg2a and igg1 antibodies were determined in wt and mif/ mice after 2, 4, and 8 weeks after stz administration . Wtstz produced high levels of igg2a at all time points analyzed (figure 4(a)) and igg1 only at week 8 (figure 4(b)). Importantly, mif/stz mice displayed significantly lower levels of both igg2a and igg1 than wtstz (figures 4(a) and 4(b), resp . ), suggesting suppressed development of an adaptive immune response . These findings suggest that mif modulates the incidence and severity of diabetes by favoring the development of autoantibodies in t1 dm . We showed that the reduced severity of t1 dm in mif/stz is associated with decreased pancreatic islet damage and diminished adaptive immune responses . To determine whether this phenotype could be related to the degree of activation of antigen - presenting cells, we characterized the expression of the costimulatory molecules cd80, cd86, and mhc - ii and of the receptors tlr-2 and tlr-4 in m and dc from the spleen and pancreas of mif/ and wt mice after 0, 2, 4, and 8 weeks of stz administration . In cells isolated from the spleen, the percentages of cd80-, cd86-, mhc - ii-, tlr2-, and tlr4-expressing m (f4/80) were similar between healthy mif/ and wt mice (figures 5(a), 5(b), 5(c), 5(d), and 5(e), resp ., at time 0 post - stz administration, right panel). Interestingly, upon stz administration, mif/ mouse m showed impaired activation, characterized by reduced expression of cd80, cd86, mhc - ii, tlr-2, and tlr-4, compared with m from wtstz mice (figures 5(a), 5(b), 5(c), 5(d), and 5(e), resp . ). Similarly, dcs (cd11c) isolated from the spleen of mif/stz mice expressed lower levels of cd80, cd86, and mhc - ii at 4 through 8 weeks after stz administration (figures 6(a), 6(b), and 6(c), resp . ), whereas the expression of tlr-2 and tlr-4 in dcs was reduced at 4 weeks in mif/stz compared to wtstz (figures 6(d) and 6(e), resp . ). To investigate the role of mif in the activation of apcs in the pancreas, we determined mhc - ii and costimulatory molecule expression in m and dc in the pancreas of mif/stz and wtstz mice . M from mif/stz expressed lower levels of cd80 and cd86 from 4 through 8 weeks after stz administration (figures 7(a) and 7(b), resp . ). Alternatively, reduced expression of mhc - ii, tlr-2, and tlr-4 was detected earlier, from 2 weeks until 8 weeks after stz treatment (figures 7(c), 7(d), and 7(e), resp . ). Moreover, dc from mif/stz mice displayed reduced cd80 at 4 to 8 weeks (figure 8(a)), cd86 at all time points analyzed (figure 8(b)), and reduced tlr-2 and tlr-4 expression at 4 weeks and 2 weeks, respectively, compared to dcs from wtstz (figures 8(d) and 8(e), resp . ). No significant differences in mhc - ii expression were detected (figure 8(c)). The ability of m to activate t cells was investigated using ova - transgenic t cell cocultures . M were collected from wt or mif/ mice treated or untreated for 8 weeks with stz . As shown in figure 9(a), after priming the cells with ova in vitro, m from mif/stz mice induced less t cell proliferation in response to ova than m from wtstz . A similar trend was observed in cocultures of cd11b cells from mif/stz and ova - transgenic t cells (figure 9(b)). These data suggest a role of mif in promoting m activation, which in turn induces specific t cell proliferation, particularly in this experimental t1 dm model . The systemic levels of mif were reconstituted in mif/stz mice during the course of t1 dm, as described in section 2 . Mif/stz that received rmif (mif/stz+rmif) displayed blood glucose levels similar to those in wtstz during the first six weeks after stz treatment . However, at 8 weeks after stz treatment, the glucose levels were not increased in mif/stz+rmif compared to wtstz (figure 10(a)). Comparable serum levels of the cytokines il-6 and il-12 were observed between mif/stz+rmif and wtstz during the first 6 weeks after stz treatment . However, the levels of these inflammatory cytokines significantly decreased in mif/stz+rmif mice at 8 weeks after stz treatment compared to wtstz (figures 10(b) and 10(d)). Interestingly, the serum levels of tnf- from mif/stz+rmif were higher than those of wtstz at all time points analyzed (figure 10(c)). These results confirm that mif acts as a powerful inducer of proinflammatory cytokines involved in the development of experimental t1 dm . Currently, there is no doubt that mif is a key molecule that promotes proinflammatory immune responses . This proinflammatory property of mif contributes to developing protective inflammatory - th1 immune response in different models of parasitic diseases . In contrast the same proinflammatory property of mif participates in the pathogenesis of many inflammatory diseases . In line with the last one, recently it has been established that high blood levels of mif are associated with human t1 dm, similar to the findings in experimental mouse models of t1 dm [20, 21, 24, 51]. Studies in nod mice or mice treated with multiple low doses of stz (despite the pathogenic differences between these models) have shown that pancreatic cell destruction results from the toxic effect of free radicals (o2, h2o2, and nitric oxide) and inflammatory cytokines released by activated m and t cells [15, 41, 52]. Therefore, both models have been widely used to dissect the role of mif in the pathogenesis of t1 dm using anti - mif monoclonal antibody treatment or using mif/ mice . In all cases, the lack of mif resulted in diminished manifestation of the disease, decreased glucose blood levels, and reduced production inflammatory cytokines associated with the development of t1 dm, including tnf-, il-1, ifn-, il-12, and il-23 [21, 22, 24]. Our study validates and extends these findings by demonstrating an important role for mif in promoting costimulatory molecule expression in m and dc during t1 dm development . We further demonstrate that, in addition to regulating m and dc activation in t1 dm, m isolated from t1 dm mif/ mice exhibit reduced t cell activation . Here, we observed that wt mice treated with stz exhibited high blood glucose levels greater than 400 mg / dl but mif/stz mice developed lower glucose levels of approximately 200 mg / dl, in association with lower serum levels of proinflammatory cytokines . After reconstituting mif using exogenous rmif, mif/stz showed blood glucose levels and il-6 and il-12 levels similar to those in wtstz from 2 to 6 weeks after stz treatment . Interestingly, tnf- serum levels from mif/stz+rmif were higher than those of wtstz at all time points analyzed . The reduction levels of blood glucose, il-6, and il-12 observed in mif/stz mice, on week 8, probably were because the animals did not receive rmif injections on week 8, as mentioned in section 2, so the residual effect of rmif from week 1 to week 7 was insufficient to induce il-6 and il-12 levels at 8 week similar to that observed in the wtstz mice in this point, at least for these two cytokines . These observations confirm that mif exogenous acts as a powerful inducer of il-6 and il-12, but only if it is present in steady high concentration . In addition, we found that mif/stz did not produce detectable levels of islet autoantibodies, in contrast to wtstz, which produced high levels of islet autoantibodies . It is well known that the early presence of islet autoantibodies is decisive in the development of diabetes by nod mice as well as humans [46, 47, 53]. Our results confirm that mif is essential for the development of hyperglycemia and suggest a role for mif not only in the innate immune response but also in the adaptive immune response in t1 dm . Recently, it has been reported that mif is produced by pancreatic -cells and that mif is released by insulin granules in an autocrine fashion [54, 55]. The chemical destruction of -pancreatic islets by stz in wt mice damaged -pancreatic islets; this condition could reduce one major source of mif in this experimental t1 dm model . However, we did not observe a reduction in mif levels in this model; in contrast, wt mice produced high serum levels of mif after stz administration . In line with this finding, it is known that the pancreatic islets remaining after treatment with stz produce high levels of mif and that an elevation of mif secretion precedes pancreatic islet death induced by ifn-, tnf-, and il-1 . This evidence establishes that stz did not influence mif production / release by pancreatic islets or other cellular sources, such as t cells, dc, and m infiltrating the pancreas . The loss of insulin production in t1 dm is related to pancreatic -cell destruction due to insulitis . We observed that wtstz developed high serum levels of mif and low insulin levels compared to mif/stz, which expressed insulin levels comparable to those in healthy mice . The histological analysis of pancreatic islets showed that wtstz displayed 100% insulitis, compared to the 33% insulitis observed in mif/stz mice . These observations confirm that mif deficiency resulted in pancreatic islet protection, probably by controlling the functional activity and modulating the secretory capacity of proinflammatory cytokines produced by t cells, dc, and m that reach the pancreatic cells . Mif has been recognized as a molecule that not only promotes proinflammatory cytokine production but also acts as a chemokine . For example, mif plays a crucial role in leukocyte recruitment and arrest during atherosclerosis development . Therefore, mif could participate in the process of insulitis to promote the production of proinflammatory cytokines, but mif could also promote leukocyte recruitment to pancreatic -cells . Antigen - presenting cells, m and dc, are key mediators of the development of t1 dm . Moreover, it has been proposed that dc orchestrate the autoimmune response in t1 dm via tlr-2 and tlr-4 . Previous studies by us and others have shown that mif induces the expression of costimulatory molecules on m and dc in some pathological infections [5961]. Here, we identified the expression of costimulatory molecules and tlr-2 and tlr-4 on m and dc, as well as the ability of m to activate t lymphocytes . Our results demonstrated that both m and dc from the spleen and pancreas of mif/stz mice expressed lower levels of cd80, cd86, mhc - ii, tlr-2, and tlr-4 than those of wtstz . These results demonstrate a role of mif in the activation of m and dc to promote costimulatory molecule expression, which might drive pancreas - specific t cell activation and effector th1 subset differentiation, processes that have been associated with subsequent pancreatic injury in t1 dm . In line with the results described above, we observed that both m (f4/80) and monocytes (cd11b) from wtstz were more reactive and had greater ability to induce t lymphocyte - specific proliferation in response to ova than those from mif/stz mice . This finding is consistent with the evidence that proliferation of antigen - specific t cells from tcr - transgenic mice is highly dependent on cd28/cd86 costimulation . This conclusion agrees with the result that blocking cd86 prevents the development of diabetes in nod mice . Moreover, mutations in the mhc - ii molecule lead to development of autoimmune diabetes . By another hand, the differentiation state of m is an important determinant for t cell response in t1 dm . Two major populations have been defined, the classically activated (ca) m secreting proinflammatory cytokines such as tnf-, il-6, and il-1 and reactive oxygen species; and alternatively activated (aa) m which secrete anti - inflammatory factors including tgf- and il-10 . In t1 dm it has been established that cam trigger inflammatory responses which initiates insulitis and pancreatic cell death, whereas the aam decreases hyperglycemia, insulitis, and inflammation in the pancreas . As mif is a regulator of many proinflammatory cytokines that are characteristic for the cam, mif has been proposed as cam macrophage - polarizing factor . However, there are few and contrary experimental evidences about how mif might participate in the polarization to aam or cam. In a mouse model of nonalcoholic fatty liver disease m in liver from mif/ mice were skewed toward aam . By contrary, in the melanoma mouse model m-derived mif participates in aam polarization . Moreover, murine mif and filarial nematode parasite (brugia) mif protein induced proinflammatory cytokines release . However, mif also induce upregulation of il-4 on bone marrow - derived mouse m, which when treated in vitro with mif and il-4 induce aam . Here, we show that m from mif/stz mice display reduced proinflammatory cytokine production and exhibit reduced ability to induce t lymphocyte proliferation in response to ova . It is possible that mif deficiency influences on aam polarization in this model; however, more experiments are necessary to establish this . We show for the first time a role of mif in promoting costimulatory molecule expression in m and dc in t1 dm . These results reveal mif as a key regulator of proinflammatory function and m and dc activation in t1 dm . Although more specific experiments are required, there is no doubt that mif represents a potential target for anti - mif therapy, which might attenuate the autoimmune process in t1 dm.
About 80% of all cancers are diagnosed at ages above 65 years, and up to 75% of cancers are thought to be associated with behavioral factors if modified, they could significantly reduce cancer burden . Analyzing an impact of the modifiable factors on cancer risk, it has been speculated that about 50% of cancers are potentially preventable . Although there are many specific results clarifying the effects of lifestyle factors on risk of lung, breast, prostate, colorectal, and other cancers, both the roles of various lifestyle factors and combined effects of multiple factors are still not clear . The availability of large datasets with more detailed information provides a new prospective in studying the role of behavioral factors in the cancer risk both for each factor alone and by taking into account risk factor interactions . The sources for obtaining the evidence on associations between behavioral factors and cancer risk include in vitro studies, animal experiments, ecological studies, and case - control studies . However, there are certain limitations in providing with exposure - to - a - factor cancer risk correlations [3, 4]. The most influential is study design biases (e.g., selection bias): for example, due to the fact that information on behavioral factors is usually collected by interviewing the patients with diagnosed cancers thus causing the bias of the estimates . The prospective cohort studies can avoid most of methodological biases; however, they are typically expensive, especially, when detailed questionnaires are required . In this paper, analysis of multiple associations between behavior factors and cancer risk is presented using the national long - term care survey linked to medicare files of service use . First, our approach is based on the cohort study in which the measurements were performed before the beginning of the cohort followup for cancer incidence . Therefore, selection or recall biases which are typical for case - control studies are not the case in the study design . Second, in earlier studies, the evaluated associations of the same type, and especially relative risks obtained for different lifestyle factors could hardly be compared between each other due to the differences in the study designs, time of measurements, and so forth . In contrast, data used in our study included multiple risk factors which were measured simultaneously, thus providing with possibility to compare evaluated associations between different risk factors . The used dataset is useful for both getting an additional knowledge about the roles of already recognized cancer risk factors as well as to establish new candidate behavioral factors which could potentially influence cancer risk in the us elderly which will provide with the background hypothesis to be tested in further analyses . Third, this study is based on population which is representative of the whole us elderly population, thus allowing to overcome such limitations of meta - analysis as heterogeneity bias, publication bias, and several others (reviewed by manton, akushevich, and kravchenko, sections 3.1 and 3.2). Two sources of data are used in the analysis: the nationally representative nltcs, for measuring functional status and behavioral factors in the elderly, and medicare claims linked to the nltcs, for cancer incidence in the us population . Breast, prostate, lung, and colon cancers were selected for analyses due to their high - incidence rates in elderly and because their incidence rates can be relatively well reconstructed from medicare data . The seer data were used as a gold standard to compare age patterns of selected cancers from seer with age patterns prediction based on nltcs - medicare data . All medicare beneficiaries receive part a benefits, which cover inpatient care in short- and long - stay hospitals, skilled nursing facilities, home health, and hospice care . About 95% of beneficiaries are also subscribed to medicare part b to obtain the benefits covering physician service, outpatient care, durable medical equipment, and home health (in certain cases). The medicare claims records contain the information on dates and costs of each service, types of providers, icd-9-cm diagnoses, auxiliary diagnostic codes, and procedure codes . The nltcs (1982, 1984, 1989, 1994, 1999, and 2004/5) contains longitudinal and cross - sectional data on a nationally representative sample of about 49,000 us individuals aged 65 + years, with 17,00020,000 age - eligible survivors in each of six rounds . The 1994 and 1999 nltcs waves were analyzed: more than 200 variables were selected in each wave of survey being grouped as follows (a complete list of all variables used in the analysis is presented in table 1 in the electronic supplementary material available online at doi: 10.5402/2011/415790): demographic characteristics (4 variables: sex, race, marital status, and urban versus rural living); self - reported comorbidity (27 major medical conditions and recent medical problems); daily living activities (22 variables: 6 activities of daily living (adls) with the two severity levels and 10 instrumental activities of daily living (iadls)); range of motion (16 variables reflecting ability to perform daily activities such as walking, using fingers to grasp and handle small objects, climbing stairs); physical activity list (29 variables, 25 of them reflecting specific physical activities (e.g., golf, tennis) were measured in 1994 only); nutrition and social activities (30 variables, 24 of them representing a nutrition survey were measured in 1999 only); alcohol consumption and smoking (4 variables, reflecting two severity levels); other functioning (28 variables reflecting self - estimates of health, information about mood, habits, keeping in touch with friends and relatives, and if they are satisfied with their life); housing and neighborhood characteristics (23 variables describing the area, housing, and amenities where sample lives, as well as including information whether he lives with other household members, and neighborhood characteristics); health insurance (6 variables containing information on coverage by medicare, hmo, medicaid, etc . ); medical providers and prescription medicine (44 variables providing with information on the use of health care services and public and private expenditures for health care services); cognitive functioning (18 variables about cognitive status of individuals, 10 of them are measured in 1994 and 11 of them are measured in 1999); income and assets (4 variables correlated with socioeconomic status of individuals); body mass index (5 variables representing body - mass index and eating style). The following concept was used for selecting the variables to be studied . First, we collected all substantive variables measured in certain nltcs surveys which were independent from responses to other questions . The most of variables were binary, and the variables with multiple outcomes were dichotomized by aggregating outcomes with similar meanings . Then, among these variables only those with low frequencies for missing data were kept: the frequencies for missing data were less than 0.02 for 65% of variables, 0.020.05: for 16% of variables, 0.050.15: for 8% variables, 0.150.25: for 8% of variables, and 0.250.45: for 2% of variables (the variables of the last group contain the questions about individual's cognitive status). For each variable (232:for 1994 and 229:for 1999 surveys) the association with four cancers incidence were analyzed using 1994 and 1999 surveys, in total, 3,688 associations . The empirical analysis and methods of univariate, two - factor, and multivariate statistical estimation with cox's proportional hazards model were used, with individual weights (so - called, cds detailed cross - sectional nltcs weights) for obtaining the us elderly population relevant results . The standard errors for all estimates were calculated based on real numbers of individuals, that is, for nonweighted populations . For small numbers of individuals in a certain stratum, corrections for the standard calculation of standard errors were used according to . For lung, colon, breast, and prostate cancers, the age - adjusted incidence rates conditional on a specific outcome (i.e., specific answer on a specific question) were estimated and the relative risks were estimated as the ratios of the rates for alternative outcomes . Note, that age - adjusted risks were calculated for subpopulations with different responses for certain questions / variables (e.g., current smokers and nonsmokers) using the same population weights for both outcomes . Therefore, the rates conditional on a specific outcome of each variable were adjusted for total population, thus taking into account a possible effect of age dependence of certain outcome prevalence . For example, lung cancer rates in smokers and nonsmokers were adjusted for age structure of total population to include smokers, non - smokers, and individuals with missing information on smoking status . Calculations of relative risks of specific outcomes for all cancers were also performed in the univariate proportional hazard model . Two basic methods of individual followups were used and compared: (1) the time - period - based followup started from the date of individual interview for which stratification by age and sex were used at the maximization of partial likelihood, and (2) the age - based followup started from the age at interview . First, individual medical histories were reconstructed from all medicare files combining all records with respective icd-9 codes: breast cancer (174.xx), prostate cancer (185.xx), lung cancer (162.xx), and colon cancer (153.xx). Second, individuals with the histories of the considered cancer before the date of interview were excluded . Then, the date of medicare record (referred as this record below in this subsection) was identified with the date of cancer onset if both two below conditions are satisfied: this record was the earliest record with respective icd code as a primary diagnosis in one of four medicare sources (inpatient care, outpatient care, physician services, and skilled nursing facilities); there was another record with respective icd code as a primary diagnosis in these four medicare sources which appeared in another claim and on a date other than the date of this record and no later than 0.3 of a year after this record . Since we analyzed the cases starting from 1994 and medicare histories were available from 1991, we had a sufficient time period (> 36 months) to reject the prevalent cases . In this analysis we also excluded the individuals with additional coverage by hmo, as well as individuals enrolled into medicare less than half a year before the interview in year 1994 or 1999 . Table 1 presents the age - adjusted rates of cancer incidence in 19941998 and 19992004 compared with those calculated for these periods using seer data . Age - adjusted estimates for associations between behavioral factors and the risk of four most common cancers (lung, prostate, breast, and colon) were calculated by three methods: (i) calculating the age - adjusted rates with adjusting of each subpopulation (i.e., with positive and negative outcomes for a specific question) for total population; (ii) based on the proportional hazard model with two approaches for choosing the follow - up variables based on age and (iii) on time . All three methods took into account the fact that age is the main and well - documented cancer risk factor . They were designed to analyze the associations for same - age individuals . Using the three approaches discussed above, we calculated the age - adjusted associations between behavioral factors and the risk of four most common cancers (lung, prostate, breast, and colon) and selected the most significant lifestyle variables associated with increased cancer risk . The strictest criterion used in the analysis was based on the bonferroni correction and an additional requirement that the found associations have to be detected both in the cohort of 1994 and 1999 . Only two associations were found to satisfy this criterion: heavy cigarette smoking and lung cancer (rr = 7), and cancer history presence (cancer site nonspecified) and breast cancer risk (rr = 6) (in part, the high relative risk could be due to a mixture of the prevalence cases which cannot be separated from incident cases). The bonferroni correction is too conservative, and it is supposed to be applied to independent hypotheses testing, which is not the case of this study due to the explanatory variables correlated especially within the specific groups . Therefore, two other criteria based on p - values equaling .05 and .002 are used . Keeping the associations with p <.05 detected at least by two of three methods resulted in a list containing 40 variables for breast cancer, 25 for prostate cancer, 43 for lung cancer, and 23 for colon cancer (see table 2 in the electronic supplementary material available online at doi: 10.5402/2011/415790). Tables 2(a)2(d) shows the sublist that included variables for which at least one of six relative risks estimated by three methods for two years has p - value lower than .002 (marked with bold font). The majority of these variables were obtained from the subgroups such as comorbidity and health status, housing and neighborhood characteristics, nutrition, social activities, and other functioning . Specific variables in the list from the other groups are body mass index and the type of insurance coverage for breast and lung cancers, alcohol consumption and physical activity for prostate and colon cancers, and cigarette smoking for lung cancer . Comorbidity is an important risk factor of cancer mortality, however its role in cancer risk is not so clear and varies depending on cancer site . Our results demonstrated that the comorbidity effect was larger for breast and colon cancers and less pronounced (but still significant) for lung cancer . Specifically, circulatory disease and certain neurological disorders increased breast cancer risk, and pulmonary diseases were associated with increased risks of lung and colon cancers . For example, having pneumonia during last year was associated with increased risk of colon cancer (rr = 2.9), and emphysema was associated with the increased lung cancer risk (rr = 3). It has been shown in other studies that prior history of respiratory diseases such as emphysema, asthma, and pneumonia was associated with increased lung cancer risk, for example, for emphysema or = 2.87 . The causal nature of the association between respiratory diseases and lung cancer remains speculative because since both emphysema and chronic bronchitis are strongly influenced by smoking . There is an evidence that inflammation may also play a role in colon carcinogenesis (through c - reactive protein and, probably, interleukin-6 factors), however, epidemiological studies are sparse . Also pneumonia could be associated with smoking, which in turn may increase the risk of colon neoplasia . At present, however, there is no proven hypothesis about the role of respiratory diseases in lung and colon carcinogenesis, and empirical evidences are not entirely consistent and are largely derived from the observational epidemiologic studies . For prostate cancer, there were indications of inverse association with comorbidity in our study, for example, prostate cancer risk was lower for persons with arthritis (both osteo- and rheumatoid arthritis) (rr = 0.48). The inverse associations for other comorbidities were not significant; however they did demonstrate the tendency . Our study also demonstrated the reduced prostate cancer risk (rr = 0.45) in patients with self - reported diabetes . These results are in agreement with recently published data from the prostate cancer prevention trial in which diabetes was associated with reduced risk of prostate cancer: or = 0.53 and or = 0.72 were detected of the risk of a low - grade and high - grade tumors, respectively . Particularly significant inverse association with prostate cancer risk (or = 0.27) was found for early - onset diabetes (diagnosed before age 30) [11, 12]. However, the mechanisms underlying these associations are still not completely clear . We have found that physical activity decreased risks of all four studied cancers, with a more significant decrease in individuals who reported moderate activities (rr = 3.5). The effect of vigorous activities was also positive (i.e., reducing cancer risk); however, the estimates of rrs varied depending on the type of physical activity . Note, that while analyzing the effects of physical activity the bias could occur due to the difficulties in measuring this factor, its overreporting, and confounding factors . However, the inverse associations with physical activity (i.e., reducing cancer risk) have been described in other studies for most of human cancers, including colorectal, breast, prostate, and lung . Our results demonstrated that maintaining normal body weight was associated with decreased risks of cancers of breast (rr = 0.55), prostate (rr = 0.6), and colon (rr = 0.4). A tradeoff between the effects of bmi (measured, not self - reported) on breast and lung cancer risks was detected: while normal bmi (1825 kg / m) reduced breast cancer risk twice, lung cancer risk increased more than three times, and vice versa, that is, bmi above 25 kg / m doubled breast cancer risk while diminished risk of lung cancer . The inverse association between bmi and lung cancer could be due to confounding smoking (i.e., smokers may maintain lower bmi easier). Data from the multiple case - control and cohort studies suggest this possibility: after adjustment for confounding smoking, the inverse association became insignificant . Other studies showed that an excessive calorie intake was strongly related to colon and postmenopausal breast cancer risk; however, not enough evidence was provided for prostate cancer risk of its association with body mass index . Two variables were used in our study to characterize alcohol consumption: (i) drinking alcoholic beverages such as beer, wine, or liquor no more than 13-times a month or not drinking at all, and (ii) consuming the alcohol at least 1 or 2 times a week . No significant associations were found for the first variable, while the second variable (i.e., heavier alcohol consumption) was associated with increased prostate cancer risk (rr = 1.7). Besides, we did not consider cancers for which alcohol consumption an evident risk factor . The effects of dietary patterns on cancer risk in our study were not statistically significant for all studied cancers, and associations for these cancers were also not proved in other studies [1720]. Specifically, no clear association was found between fruits and vegetables consumption and reduced colon cancer risk . These results do not support the existence of protective role of dietary fiber against colon cancer [21, 22]. Also, no association was found in our study between beef, pork, and lamb (without specification on well - done or other cooking regimen) consumption and increased colon cancer risk . The recent meta - analyses showed that high intake of processed meat but not fresh meat could increase risk of colon cancer [23, 24], while well - done meat could increase colon cancer risk in susceptible individuals with rapid - rapid phenotypes of nat2 and cyp1a2 . No associations have been found in our study between breast, colon, and lung cancer risk and different levels of disability . This is in accordance with the results obtained from other studies where no significant protective effects of disability were found . However, a markedly decreased risk of breast cancer was observed among disabled older women compared with physically capable but inactive women . In our study these associations are of noncausal character and can be further investigated in two - factor analysis using other measured variables as confounders or mediators for their explanation . They could also be due to unobserved heterogeneity in cancer risk and could potentially be clarified in future studies . An example of such an association is the relationship between breast cancer risk and nine variables from the hnc group (e.g., positive responses to questions like which of these things would make things easier or more comfortable for you: extra wide doors or hallways, push bars on the door, extra handrails, etc?) Which are strongly associated with breast cancer risk with rr from 4 to 7 and p - value of the association less than .001 . No associations of these variables were found for risks of other cancers . Because of occurrence of false - positive results while testing the hypotheses and/or noncausal nature due to observed and unobserved confounding, the second step in the analysis was the two - factor analysis including effects of interactions between risk factors allowing for revealing effects of confounding and effectively taking into account the mutual correlations inside the groups of similar questions . Analysis of simultaneous effects of two variables allowed us to check whether associations found in unidimensional analyses were confounded by other measured variables . Simultaneous effects of all possible pairs of variables were evaluated using the cox proportional model focusing on detecting the significant change in the estimated relative risk (or the loss of the significance of the estimate) after adding the second variable . Several types of the effects of second variables were identified: confoundings, candidate mediators, independent predictors, and overlapping predictors (notation is discussed by [28, 29]). As expected, smoking was the strongest and most often confounding of other risk factors for lung cancer: it changed substantially the effects of sex (by 2.2 e, where e is the standard error of the rr estimate in unidimensional analysis, in 1994 and by 0.6 e in 1999), urban living (0.8 e), easy loosing temper (1.2 e), emphysema (0.7 e), and bmi (0.7 e) on lung cancer risk . The bmi changed effects of diet (about 1.0 e) and lost appetite (0.6 e). Smoking, physical activity, social activity, satisfaction with life, overeating, type of medical insurance, and access to medical services (except of the veteran administration insurance) were independent from mediating lung cancer risk by other factors . Certain variables whose effects changed the initial effect of independent variable (i.e., that in unidimensional analysis) can be causally linked to the effect of independent variable; therefore, they can be considered as mediators . Detailed analysis of such causal pathways requires further investigation using a theory of statistical mediation mackinnon and will be performed elsewhere . For other cancers, the bmi influenced effects of certain comorbidities (such as circulatory diseases, about 1.0 e) and overeating (0.6 e) on breast cancer risk, while the effects of vitamins, social activities / hobbies, and contacts with relatives were independent from the effects of other . For colon cancer, bmi, being almost an independent factor, mediated the effects of sex (1.4 e), alcohol consumption (0.8 e), and overeating (0.7 e). The two - factor analysis also revealed the situations with overlapping effects among variables which are correlated, codominant, and have no temporal precedence, for example, effect of variables from the hnc group on breast cancer risk . This is because of their mutual correlation and the so - called effect of statistical collinearity, when the estimated effect of a predictor cannot be interpreted itself . Summarizing, the effects of confounding evaluated in two - factor analysis do not change the conclusions made while using univariate approach but further specify the evaluated associations . Also this analysis clarified that further progress can be achieved by investigating the combined effect of correlated variables (e.g., those from the same nltcs group) by constructing an aggregated index . In this study, we analyzed how lifestyle factors represented by a number of variables were associated with incidence of four most prevalent cancers such as lung, prostate, breast, and colon . Overall view on the results of association analyses allowed us to describe population groups of higher and lower risks of these cancers . Being a smoker was the main characteristic of elderly population group of higher risks of lung cancer, with comorbidity (e.g., emphysema), lower bmi, and poor functional status also each playing the role . The population of higher risk of colon cancer was characterized by a higher bmi and comorbidity . The elderly women at higher breast cancer risk reported higher occasional activity and intentions to improve things around her day to day (these relationships could be indirect and require further investigation). The group of higher risk of prostate cancer had lower comorbidity, disability, and functional status (partly, it could be due to the underdiagnoses in individuals with poor health state). In this study, many well - recognized associations were confirmed; however, certain fundamental questions about lifestyle effects on cancer incidence remain unclear . Specifically, directions for further investigations could include analyses (i) of comorbidity variables for which the inverse associations with prostate cancer was found (e.g., arthritis or diabetes), (ii) of associations indirectly related to cancer risk variables (e.g., housing, neighborhood, or income characteristics) which could be potentially explained in terms of confounding factors . From biomedical perspective, potential extension of this study could include the nltcs - medicare data analysis clarifying (i) how found cancer site - specific associations could be affected by racial disparities and (ii) whether there is a difference in factors effects and their mediation for cancers of reproductive and nonreproductive systems as well as more detailed analysis of sex - specific associations and potentially different role of certain factors in mortality and cancer risk in males and females . Further analysis could deal with implementation of interactions between two or more variables using multi - factor analysis, applying the theory of statistical mediation, searching for so - called instrumental variables, and constructing quasirandomization using propensity score approach (reviewed by faries et al . ). For example, the propensity score can be evaluated for each association by considering respective independent variable as exposure or treatment and using all other measured variables as predictors of the exposure in logistic regression . Another further investigation could focus on searching the latent variables capable of describing the heterogeneity in cancer risk: for example, for lung cancer such a variable could be self - care, psychological condition, or happiness (the latter is also a candidate variable for breast cancer). One formal method capable of identification of the latent variables associated with certain risk is the linear latent structure analysis [31, 32] when a score is identified by statistical methods and analyzed in a certain basis each component of which is associated with a group of higher / lower risk . Important feature of the method is that it takes into account mutual correlation between predictors . The most influential (i.e., demonstrated the strongest association with cancer risk) of potentially controllable risk factors can be detected using the approach developed in this paper and then applied to further deeper analyses, including other data sets with detailed risk factors description / characteristics, for example, analyses of duration of exposure and intensity of risk factor . These approaches could provide with the steps toward the individualized forecasting of cancer risk potentially resulting in preventive strategies which could be oriented to population groups with specific characteristics such as those obtained from the indices and association / confounding findings of this study.
Even though substantial efforts have been made to improve education and public awareness and despite the use of effective medications and life - style changes for controlling the associated risk factors, coronary artery disease (cad) remains the leading cause of death in women worldwide [1, 2]. In contrast to age - matched men, the incidence of clinical manifestations of cad is considerably lower in premenopausal women; however, most of women develop cad after menopause when endogenous estrogen levels are low [35]. During normal menstrual cycles, women show high levels of estrogen just before ovulation and during the luteal phase and in the normal physiology of pregnancy, women have significantly higher levels of estrogen derived mainly from the placenta . Estrogens have been known to exert various positive effects on the cardiovascular system [7, 8]. It has thus been shown that estrogens retard the atherosclerotic process and induce rapid vasodilatation through the production of an endothelium - derived vasoactive mediator, nitric oxide (no) [810]. Hashimoto et al . Reported that endothelium - dependent vasodilatation is increased in young women during the phases of their menstrual cycles when endogenous estrogen levels are high, and pregnant women show significantly high levels of estrogen . Some studies have documented that estrogens are potent antioxidants and decrease low - density lipoprotein cholesterol (ldl - c) oxidation in vitro and in vivo [12, 13]. While estrogens decrease lipid peroxidation and formation of reactive oxygen species, androgens and progestins increase oxidative stress parameters . Lectin - like oxidized low - density lipoprotein receptor-1 (lox-1), a type ii membrane glycoprotein, is the major receptor for oxidized low - density lipoprotein (ox - ldl) in endothelial cells . Oxidative stress and ox - ldl both alter endothelial biology by activating a specific receptor lox-1 . The activation of lox-1 has been shown to lead to further oxidative stress in endothelial cells and the appearance of proinflammatory phenotype . Lox-1, furthermore, is cleaved at the membrane - proximal extracellular domain by proteases [18, 19] that may also be associated with endothelial dysfunction and atherosclerotic plaque formation and destabilization, resulting in soluble lox-1 (slox-1) release into the circulation . Since the level of soluble receptors in circulating blood may reflect the expression of membrane proteins and disease activities, slox-1 may be a potential biomarker of vascular disease assessment . Therefore, we hypothesized that if women have been exposed for a longer time and/or at a higher level to endogenous (not exogenous) estrogen, such as pregnancy followed by delivery and/or gravidity, they may obtain estrogen's beneficial the purpose of the current study was to determine the association between pregnancy followed by delivery and slox-1 . From january 2010 to june 2011, we prospectively evaluated 1284 patients in cardiology outpatient clinic of our hospital . Sixty - eight subjects with pregnancy followed by delivery (group 1) and 57 subjects with nongravidity (group 2) were included in this study . Exclusion criteria included pregnancy, known polycystic ovary syndrome, congestive heart failure (ejection fraction <50%), myocardial infarction, stroke, known peripheral atherosclerotic disease, surgical coronary intervention, other major vascular surgical procedures, coronary angioplasty, unstable angina pectoris, diabetes mellitus, hypertension, suspected myocarditis or pericarditis, impaired renal function (creatinine 1.4 mg / dl), unstable endocrine or metabolic diseases known to influence serum inflammation markers, concomitant inflammatory diseases such as infections and autoimmune disorders, active or chronic hepatic / hepatobiliary disease, and malignancy . Patients taking oral contraceptive, corticosteroids, anti - oxidant vitamins, and alcohol were also excluded from the study . Blood samples of all individuals were taken from an antecubital vein following an overnight fasting state at the first three days of menarche . After centrifugation at 3000 g for 10 minutes, serum and plasma samples were frozen and stored at 80c until an assay could be performed . Serum slox-1 levels were measured by a commercially available enzyme - linked immunosorbent assay kit (uscn life science, wuhan, china). The detection limit for serum slox-1 level was 2.4 pg / ml with a coefficient of variation <5% . Triglyceride (tg), total cholesterol (tot - c), ldl - c, and high - density lipoprotein cholesterol (hdl - c) concentrations were measured by automated chemistry analyzer (roche diagnostics, indianapolis, usa) by using commercially available kits . Continuous variables were given as mean sd; categorical variables were defined as percentages . Comparisons between group-1 and group-2 were carried out using an independent samples t - test . Spss 15.0 software was used for basic statistical analysis (version 15, spss inc ., and chicago, il, usa). A value of p <0.05 was accepted as statistically significant . The mean age was 33.5 6.1 years in pregnancy followed by delivery group and 35.5 7.5 years in nongravidity group (p = 0.1). The mean age of first menarche was 12.1 2.3 years in pregnancy followed by delivery group and 11.9 1.9 years in nongravidity group (p = 0.5). The rates of family history and smoke were similar between the two groups (table 1). The smoker subjects in group 1 had 3.5 1.3 pack - year history of smoking, and smoker subjects in group 2 had 5.0 2.1 pack - year history of smoking (p = 0.1). The levels of total - c, ldl - c, hdl - c, and triglyceride were also similar between the two groups (table 1). The slox-1 levels were significantly higher in nongravidity group than pregnancy followed by delivery group (0.78 0.13 ng / ml and 0.52 0.18 ng / ml, resp ., the slox-1 levels highly negatively correlated with the number of gravida (figure 2, r = 0.645, p <0.001). Figure 3 shows a highly negative correlation between slox-1 levels and number of parous (r = 0.683, p <0.001). The slox-1 levels were not correlated with age and age of first menarche (r = 0.055, p = 0.541 and r = 0.015, p = 0.865, resp . ). In the multiple linear regression analysis age was positively related and number of gravity was negatively related with slox-1 levels (for age p = 0.011, beta = 0.169, t = 2,589 for parous p to the best of our knowledge, this is the first study that shows the relationship between slox-1 levels and pregnancy followed by delivery in women of reproductive age . This study showed that women who had at least 1 pregnancy followed by delivery showed a decreased level of slox-1 compared with those who had never experienced delivery . We hypothesized that if women have been exposed for a longer time and/or at a higher level to endogenous (not exogenous) estrogen, such as pregnancy followed by delivery and/or gravidity, they may obtain estrogen's beneficial effect and may have a greater decrease in level of slox-1 . These findings may support the idea that as long as women are exposed to endogenous estrogen they have decreased level of slox-1 . Coronary artery disease remains the leading cause of death in the 21st century . Despite the advances in this area, the prevalence of cad in premenopausal women is smaller than in postmenopausal women, when there is an exponential increase, making the risk for women equal to that for men by the age of 6570 years . This lag concerning the age period at which the frequency of cardiovascular events increases among women as compared to men has been ascribed to the actions of endogenous estrogen on the cardiovascular system, through mechanisms as yet not completely clarified . The well - known risks for cad, such as systemic hypertension, smoking, obesity, sedentary life - style, dyslipidemia, stress, family history of cad, diabetes mellitus, menopause, lack of endogenous estrogen, and insulin resistance, are numerous . The term endothelial dysfunction is more frequently used to refering reduction in endothelium - dependent vasodilatation, associated with diminished bioactivity of local vasodilative factors (especially no). Data from prospective trials have been confirming the hypothesis that endothelial dysfunction precedes the emergence of chronic disorders . Currently, it is a consensus that endothelial dysfunction is the initial event in development of atherosclerosis . There are many techniques for investigating the endothelial function, from those that focus on cellular and molecular aspects, through methods involving tissue culture and molecular biology tools, to clinical trials applied to human beings, using invasive and noninvasive procedures to evaluate endothelium - dependent vasodilatation, or the determination of plasmatic substances that indicate endothelial activation and/or damage . The incidence of cad and mortality is very low in women of reproductive age but rises to a significant level in menopause women . There is evidence of an association between endothelial dysfunction and reduced endogenous production of estrogens after natural or surgical menopause or premature ovarian failure in women with or without cad [2427]. The actions of endogenous estrogens on the cardiovascular system can be mediated directly on the vessels or indirectly through the modulation of cardiovascular risk factors, as well as on the lipid profile . The direct effects of estrogen on the vascular system and which modulate the vascular tonus comprise the following 1 acute vasodilatation, increasing the synthesis and bioactivity of no [29, 30]; 2 long - term modulation of vascular tonus, regulating the production of prostaglandins and expression of endothelial nitric oxide synthase and the endothelin gene; 3 inhibition of endothelin - induced vasoconstriction; and 4 inhibition of sympathetic activity . In addition to these actions on the vascular tonus, estrogen exerts an antiproliferative action on the vascular smooth layer . Also, it appears to have a major role in vascular remodeling, inhibiting the proliferation of the inner layer after injury and increasing the expression of contractile proteins in the myocardium . Disturbances in endothelial function have an important role in the physiopathology of atherosclerosis, and several lines of evidence suggest that interventions in endothelial function could modify the progress rates of atherosclerotic disease and the risk of cardiovascular events . Some studies have documented that estrogens are potent antioxidants and decrease ldl - c oxidation in vitro and in vivo [12, 13]. Studies on the mechanism of estrogen antioxidant effects have shown that estrogen strongly inhibits superoxide formation with minor effects on hydrogen peroxide and hydroxyl radical formation . While estrogen decreases lipid peroxidation and formation of reactive oxygen species, androgens and progestins increase oxidative stress parameters . Clinical studies on humans using 17-estradiol - based preparations have clearly shown decreased ldl - c oxidation, and in addition, estradiol reduces the development of early lesions of atherosclerosis, in part through the effects on lipid metabolism which reduce lipid deposits in the endothelium [36, 37]. Demonstrated that a high concentration of estrogen reduces the level of asymmetric dimethylarginine (adma), which is an endogenous competitive inhibitor of no synthase . Hashimoto et al . Also demonstrated that women who had had at least 1 pregnancy followed by delivery showed a decreased level of arteriosclerosis, measured noninvasively by brachial - ankle pulse wave velocity (ba - pwv) as an indicator of arteriosclerosis . It was closely correlated with aortic arterial stiffness and the severity of atherosclerosis, compared with those who had never experienced delivery . Human umbilical vein endothelial cells exposed to high concentration of 17-estradiol were used as an antiatherosclerogenic agent to demonstrate feasibility in an in vitro vascular model . Lox-1, a type ii membrane glycoprotein, is the major receptor for ox - ldl in endothelial cells . Oxidative stress and ox - ldl both alter endothelial biology by activating a specific receptor lox-1 . The activation of lox-1 has been shown to lead to further oxidative stress in endothelial cells and the appearance of proinflammatory phenotype . Lox-1 has been implicated in vascular inflammation and atherosclerotic plaque formation, progression, and destabilization [42, 43]. Lox-1, furthermore, is cleaved at the membrane - proximal extracellular domain by proteases, including a disintegrin and matrix metalloproteinases (mmps) [18, 19] that may also be associated with plaque vulnerability or rupture, resulting in soluble lox-1 (slox-1) release into the circulation . In addition, plasma slox-1 levels were higher in males and smokers than in females and nonsmokers, probably because endogenous estrogen and smoking affect plaque vulnerability by protecting vascular cells from inflammation (the former) and by inducing oxidative stress and inflammation (the latter). In experimental animal models, lox-1 expression is closely associated with morphological plaque instability and cell apoptosis, as well as with the expression of mmps and tissue factor, all of which are associated with plaque rupture and thrombus formation [4547]. A study demonstrated that lox-1 deficiency significantly decreases the formation of atherosclerotic lesions and endothelial dysfunction . It is well known that menopause or lack of endogenous estrogen is a risk factor for cardiovascular disease [4951]. Hashimoto et al . Reported that women who are regularly menstruating have a decreased pwv compared with post - menopausal women of the same age and a younger age at menarche correlates with pwv reduction . This finding may support the idea that as long as women are exposed to endogenous estrogen they have decreased endothelial dysfunction . Thus, ovarian dysfunction and either natural or surgical menopause have been recognized as a major risk factor for accelerated atherosclerotic vascular disease development [3, 52]. In stages of disrupted ovulatory cycling, low levels of endogenous oestrogens during premenopausal years accelerate the progression of atherosclerosis [53, 54], which can be reversed by oestrogen therapy in animals . In addition, results from experimental studies and recent clinical trials indicate that oestrogen therapy started within few years after menopause, that is, before the development of severe atherosclerosis, may in fact reduce cardiovascular risk [5, 5559]. In contrast, initiation of oestrogen therapy many years after menopause, that is, when advanced and multiple atherosclerotic lesions are present, may have no or even deleterious cardiovascular effects [5, 5559]. In conclusion, our study demonstrated that serum slox-1 levels were associated with pregnancy followed by delivery which might predict endothelial dysfunction . Pregnancy followed by delivery may improve endothelial function and prevent the progress of atherosclerosis in women of reproductive age . We conclude that pregnancy followed by delivery may delay the progress of arteriosclerosis and its clinical manifestations in women of reproductive age.
Dissemination and implantation of gastrointestinal malignancies throughout the peritoneal cavity result in peritoneal carcinomatosis (pc), which is the second most frequent cause of death in colorectal cancer after metastatic disease to the liver . In 25% of colorectal cancer cases, the peritoneal cavity is the only site of metastatic disease . Despite recent advances in the development of systemic chemotherapeutic agents, pc attempts have been made in the past decades to increase long - term survival in patients with pc by combining cytoreductive surgery (crs) to remove visible disease and hyperthermic intraperitoneal (ip) chemotherapy (hipec) to eradicate microscopic viable residual disease, with elevated and persistent drug concentration in the peritoneal cavity . The peritoneal route of chemotherapy administration has the advantage of delivering the agent to the entire peritoneum after all adhesions have been lysed and before they have the opportunity to reform . The peritoneal - plasma division concept allows a tenfold higher concentration of the chemotherapy to be reached in the abdominal cavity in direct contact with residual cancerous cells while minimal systemic absorption and adverse effects are expected . The addition of hyperthermia results in a potentiation of cytotoxicity, improving its capability of penetration into tumoral masses and serving to rewarm the patient after a significant open procedure . The ideal ip chemotherapeutic agent should be one that has maximal efficacy, offering optimal regional therapeutic benefits, while minimizing systemic toxicity . Mitomycin c (mmc) has been the most frequently used ip chemotherapy for gastrointestinal malignancies . The high molecular weight of mmc limits its systemic absorption and toxicity after ip administration . More recently, based on the efficacy of new systemic agents for patients with metastatic colorectal cancer, oxaliplatin and irinotecan have emerged as good options for peritoneal perfusion [6, 7]. Although there are no randomized trials comparing mmc with oxaliplatin as ip chemotherapy, a retrospective study with 539 patients with pc associated with colorectal cancer showed that both agents achieve similar median overall survival (32.7 vs. 31.4 months; p = 0.925). However, due to shortage of mmc in brazil, we have been using oxaliplatin as the preferred agent . Despite all those potential benefits associated with crs with hipec, morbidity and mortality rates are substantial . Large series of patients undergoing crs with hipec report mortality rates ranging from 1 to 8% [9, 10, 11, 12, 13]. In the only randomized study (n = 105) comparing systemic chemotherapy (fluorouracil - leucovorin) with or without palliative surgery with aggressive crs with hipec, followed by the same systemic chemotherapy regime, the mortality rate in the crs with hipec group was 8% . In addition, surgical morbidity, such as postoperative ileus, anastomotic fistula, and wound infection, as well as medical morbidity, including pharmacological toxicity, cytopenia, bone marrow aplasia, renal toxicity, and hydroelectrolytic disorders, are quite frequent following crs with hipec [3, 9]. The most common reported complications include enteric fistula, intraabdominal abscess, pneumonia, small - bowel obstruction, pancreatitis, and neutropenia which often prolong the hospital stay . To the best of our knowledge,, we describe the case of a previously healthy young man who underwent crs with hyperthermic ip oxaliplatin and developed one episode of tonic - clonic seizure on the second postoperative day . A 26 year - old man presented in mid-2011 with increasing abdominal pain . In october 2011 he was found to have a poorly differentiated adenocarcinoma of the transverse colon with multiple peritoneal implants . It was consistent with a pt4 pn2 (4/28) pm1 colonic adenocarcinoma, kras wild - type . From january to july 2012, he received 12 cycles of folfiri (folinic acid, 5-fluorouracil, and irinotecan) plus bevacizumab at another institution and achieved complete clinical response . At that time a pet - ct scan revealed lesions with increased fdg uptake in the peritoneum as well as in the colonic anastomosis . From june 2014 to june 2015, he received 24 cycles of folfiri and cetuximab with initial response followed by stable disease . In september 2015, he was first seen at our institution and, during a multidisciplinary meeting, it was decided to pursue crs with hipec (oxaliplatin 300 mg / m), which was started on october 5, 2015 . On the second postoperative day, the patient developed one episode of generalized tonic - clonic seizure which lasted for approximately 2 min . He was started on phenytoin to prevent new episodes of seizures during the postoperative period . On october 15, due to no recurrence of seizures, the anticonvulsant was discontinued . He stayed at the icu for only 72 h and had the first bowel movement 6 days after surgery . On the 7th postoperative day, he developed fever with no identified origin and was started on piperacillin - tazobactam and vancomycin . At this time, the patient was also complaining of left scrotal pain and swelling . An ultrasound of the testis was performed on october 17 and showed normal testicles and increased volume of the left epididymis with heterogeneous echogenicity . In addition, analysis of the epididymal waveform revealed a low - resistance pattern suggestive of acute epididymitis . Despite all cultures having remained negative, it was decided to switch antibiotics to amikacin for 10 days and to doxycycline for 14 days and treat the symptoms as infectious epididymitis . The patient was discharged home on october 22, 2015, to complete antibiotics at the outpatient unit . . Ten percent of adults experience a seizure some time during their life . Each year, 300,000 people experience a first epileptic seizure; most of them are younger than 18 years old . Seizures result from a shift in the normal balance of excitation and inhibition within the central nervous system as well as from abnormal brain function . Studies indicate that 2530% of first seizures are acute symptomatic or provoked by a brain insult or a metabolic / toxic disturbance of brain function . Provoking factors include fever, head injury, excessive alcohol intake, withdrawal from alcohol or drugs, hypoglycemia, electrolyte disturbance, brain infection, ischemic stroke, intracranial hemorrhage, and proconvulsive drugs (such as clozapine, maprotiline, tramadol, theophylline, and baclofen). There are several alterations in the physiology of patients during crs and the administration of hipec . The body temperature increases significantly due to hipec, they experience transient hyperglycemia secondary both to stress from surgery and hyperthermia, as well as the presence of dextrose in the perfusate . However, none of those abnormalities has ever been reported to cause seizures, as seen in our case . There are no published articles regarding neurological complications such as seizures in patients who were submitted to hipec with oxaliplatin . There is, however, one case report of a 37-year - old female patient with a previous history of seizure disorder who underwent crs and hipec with mmc without intraoperative complications and developed signs and symptoms of cerebral edema 4 h after the operation was completed . She eventually died of cerebral edema, with no established etiology . Despite a normal eeg, it was hypothesized that crs with hipec could have caused unrecognized partial complex seizures leading to acute cerebral edema . Even though our patient had no history of seizure disorder, it is a possibility that the seizure threshold may have been lowered secondary to the stress of surgery and hyperthermia . In fact, seizures are a rare side effect of intravenous oxaliplatin, which are usually associated with posterior reversible leukoencephalopathy syndrome (pres). Pres is characterized by neurologic symptoms such as headache, altered mental status, visual disturbances, and seizures with typical lesions on neuroimaging shown as bilateral, subcortical, symmetric, and vasogenic edema . However, our patient had no symptoms or radiologic findings of pres . There is a published case of a previously healthy man with mixed adenoneuroendocrine carcinoma who had seizures after the third and fourth cycles of intravenous folinic acid, 5-fluorouracil, and oxaliplatin (folfox) with no signs of pres . After the chemotherapy regimen was switched to folfiri, he never had another event . In this case, the authors attributed the seizures to the intravenous oxaliplatin . Our patient had no electrolyte abnormalities, no signs of infection, brain structural alteration, or other metabolic disorder that could possibly explain the single episode of seizure . Therefore, it may be possible that the seizure was caused either by the physiological modifications that occurred during hipec or by ip oxaliplatin . Because of the increasing use of crs with hipec as an option to treat selected patients with pc, it is important to report those postoperative complications . This article reports an unprecedented case of seizure in a previously healthy patient who underwent crs with ip oxaliplatin due to metastatic colon adenocarcinoma . Despite this being a rare case, the corresponding author acknowledges that she is responsible for complying with ethical requirements and declares that the patient was correctly informed, and written informed consent was obtained; the confidentiality of the patient was strictly preserved . The patient was informed about the submission of the manuscript and will be acquainted when the article is published.
It has been estimated that there were 7.6 million fatal cases of cancer (13% of all deaths) and around 12.4 million new cancer cases in the year 2008 worldwide . Deaths from cancer are forecasted to continue to grow to over 13.1 million in 2030 . The earlier the cancer is detected, the better the chances of the patient recovering are, as appropriate treatment can be applied in time . There are two components of efforts to detect cancer early: early diagnosis and screening . However, there is a lack of characteristic early clinical symptoms in most cancer types that could lead to early detection of the disease [25]. In addition, cancer diagnosis often requires many tests, some of which are invasive surgical procedures . For example, a new, noninvasive method of lung cancer screening, spiral computer tomography, which has been shown to detect cancer that is curable by surgery, is also accompanied by a risk of exposure to radiation, high false - positive rates, and the possibility of overdiagnosis . This underlines the need for investigation of new methods for the early detection of cancer . In this search all omics approaches (genomics, proteomics, and metabolomics) have been applied [79]. One of the most promising metabolomic approaches is the analysis of volatile organic compounds (vocs), which could potentially serve as a safe, noninvasive (at least for breath and some biofluid samples), and specific test for the early detection of different types of cancer . Vocs are a diverse group of carbon - based chemicals that are classified on the basis of their retention time and boiling point (ranging from 50c to 260c). Vocs are emitted from the body in exhaled breath and are present in body specimens such as blood, urine, faeces, and sweat [1114] and therefore may be collected from the headspace (hs) of these matrices, but also from the hs of cells in vitro . Different patterns of vocs have been correlated with various diseases and syndromes such as cancer, asthma, cystic fibrosis, diabetes, tuberculosis, chronic obstructive pulmonary disease, heart allograft rejection, and irritable bowel syndrome . These correlations are based on the hypothesis that pathological processes, occurring as a consequence of disease, can generate new vocs that the body does not produce during normal physiological processes and/or alter the concentrations of vocs . These new vocs, or vocs that are produced in significantly higher or lower levels than normal, may therefore serve as biomarkers for the assessment or detection of disease . This review firstly discusses sample matrices that were used in the studies of potential voc biomarkers of cancer and critically evaluates in vitro and in vivo approaches applied in this field . The investigation of targeted vocs only (rather than all the vocs present in a sample) as candidate cancer biomarkers is also discussed . Next this paper reviews complementary in vivo, ex vivo, and in vitro studies conducted to date in order to find volatile biomarkers of cancer . Finally, the main extraction techniques and analytical techniques that have been applied to date in the area of the studies of potential volatile biomarkers of cancer are compared . In order to investigate vocs as cancer biomarkers, analysis of the exhaled breath of patients with different types of cancer has become very popular in recent years [23, 24]. Alternative approaches include the hs analysis of cancer cells, tissues, or body fluids . Studies have shown that chemical changes in blood due to the presence of cancer are echoed in an alteration of the composition of vocs in the breath of patients [25, 26]. Therefore, it is hypothesised that abnormal vocs produced by cancer cells are discharged via the blood stream into the endobronchial cavity and finally exhaled with breath . Breath analysis, compared to blood and urine tests, is noninvasive and a sample may be easily collected at any point and in varying quantities, which makes it easy to repeat . Furthermore, it does not require special storage conditions or any further work after collection . In addition, the breath matrix is a less complex mixture than urine or blood . Different vocs were detected in the breath of 50 people, and only 27 were found in the samples of all the subjects . Approximately half of these 3500 compounds are of possible endogenous and half of possible exogenous origin . Only compounds produced inside the body can be considered as biomarkers, which is problematic as the origin of most volatile metabolites is still unknown or remains the subject of speculation [27, 30]. The presence of both endo- and exogenous vocs in exhaled air is one of the biggest limitations of breath analysis . The majority of the detected vocs were found only once in one particular individual and the patterns of vocs may change according to food consumption, smoking, gender, age, and so forth [31, 32]. There are different opinions about how detailed knowledge is required for a successful breath diagnostic test . Some argue that there is no need to know the origin of a volatile compound biomarker, as long as it can be used to distinguish disease from a healthy state [33, 34]. Others simultaneously measure exhaled and inspired air since the environmental contaminant vocs may be incorrectly assigned as endogenous compounds . Finally, the last approach requires knowledge about the metabolic pathway of the compound, as well as about normal concentration ranges of a compound in relation to interindividual variability, before including it into the predictive model of the disease . Moreover, since the beginning of breath analysis in the 1970s standardisation and reproducibility of the sample collection method have been an issue which has resulted in the variability of quantitative information [37, 38]. Standardisation is easier to achieve for serum or urine than for breath collection, which is a big advantage of these matrices . Furthermore, equipment for exhaled breath collection is relatively expensive and may thus not be easy to apply widely . The importance (limitations and/or applications) of breath analysis has been described previously [30, 31, 3742]. Although vocs detected in blood and urine are in the body analytes, it still does not mean they are of endogenous origin . Some inhaled vocs may bind to or dissolve in blood and be stored in body compartments and later excreted through urine . In addition, it is not known which volatile compounds are produced or consumed by tumour cells as they may also be generated (or consumed) by noncancerous cells (such as surrounding tissue cells or other regions of the body) [45, 46], immune - competent cells, human symbiotic bacteria [48, 49], and infectious pathogens [50, 51]. Furthermore, voc patterns differ between individuals because of uncontrolled variables such as genetic differences, environmental settings, diet, drug ingestion, and smoking [31, 32], which makes voc analysis a challenge regardless of the matrix used . Nevertheless, there is growing evidence that vocs that are potentially clinically relevant may be found in breath and other matrices . Dogs were reported to discriminate between patients with or without cancer by sniffing skin, blood, urine, or breath samples of cancer patients, which suggests that characteristic voc signatures of cancer exist [5257]. Sensor mice were also trained to distinguish mice with experimentally - induced cancer from mice without it . Blood was used as a matrix for voc collection in a number of studies of lung cancer [26, 59], childhood forms of cancer, and liver cancer . The disadvantages of blood as a matrix include invasiveness, and careful handling and further work after collection as temperature and ph changes can alter voc profile [37, 62] when there is a necessity to collect many of such samples, breath analysis would be a better alternative, especially as it closely mirrors the arterial concentrations of metabolites . In theory, the composition of volatile compounds in breath is related to the composition of these compounds in blood [23, 26]. This needs to be addressed in studies comparing voc composition in blood and breath samples . The study showed that 23 vocs found in blood were also present in the exhaled breath of lung cancer patients . Hexanal and heptanal were detected only in cancerous blood and breath samples and were not found in controls . However, more study is required to compare voc patterns in both matrices, where ideally the blood and breath samples from the same patient would be investigated . Many studies have also investigated volatile biomarkers in urine samples of patients with various cancers such as breast, gastroesophageal, lung, leukaemia, colorectal, lymphoma, childhood leukaemia, and bladder cancer . In addition to its noninvasive nature and availability in large volumes, urine as a matrix for voc analysis also has an advantage over other biofluids in that analytes are concentrated by the kidney before being excreted from the body . In addition, when compared to blood, the use of urine usually results in better detection limits as matrix effects may interfere with the release of the vocs into hs in blood sampling . On the other hand, vocs in urine may be affected by the drugs administered to a patient, and therefore the metabolic products of particular changes must be known as well as determining their effect on the vocs produced . The investigation of vocs produced by cancerous cells in the microenvironment as the source of biomarkers should hypothetically help with the dilemma of their origin, as advantages of in vitro studies over other matrices include easier control of experimental variables and more easily interpreted results, due to the absence of factors such as gender, age, and interindividual variation (with the exception of primary cell cultures). However, this matrix still does not guarantee that the collected vocs are of endogenous origin . They may not be produced by cancer cells themselves and may instead come from other sources such as culture vessels, extraction devices, and the sampling environment [69, 70]. The cell metabolome is comprised of the endometabolome, which is represented by all metabolites inside the cell, and the exometabolome, which is made up of all metabolites present in the extracellular cell culture medium . The profile of these metabolites in the surrounding medium depends on the uptake and extraction of the compounds by the cells and reflects their metabolic activity via their response to experimental variables . In vitro studies aiming to find potential volatile markers of cancer essentially apply the extracellular metabolite investigative approach . Endometabolomic studies require cell disruption, and then concentration of the extracted compounds (mainly with the use of evaporation). A number of studies have been performed to investigate potential voc cancer biomarkers in vitro in different types of cancer and using different techniques, and in all of them there were differences observed in the composition of volatile metabolites produced by cancer and normal cells [6981]. However, some studies found differences in voc levels, or vocs produced, between not only different cell lines of the same cancer (showing that their metabolic pathways are different) but also the same cell line [15, 75, 7982]. While the first observation may be explained by genetic and phenotypic differences and the fact that each cell line is representative of only a small part of a primary tumour, the reasons for the second are unclear [15, 80]. It may be due to the cell line being subcultured a different number of times . The study of sponring et al . Showed the possibility of a change of released volatile metabolites with increasing passage number . Cells should not be subcultured for a long period of time to ensure they have not mutated, as mutation could cause them to no longer reflect the properties of the tumour of origin . The fact that there were significant experimental differences in many studies between the cell cultures that had been subcultured a low number of times, compared to those that had been subcultured a high number of times, and the fact that there were studies conducted on cross contaminated cell lines make a compelling case for the use of certified cell lines with defined passage numbers . In the cell / tissue hs analysis of vocs there are also differences in the techniques used, and a lack of standardisation and normalisation of the data, even when the same technique is used, which may influence variations in voc patterns between different studies . The aspects to be considered (apart from the technique used) (table 1) in terms of in vitro studies of vocs include the analysis of different matrices, the use of different cell culture media, the period of cell cultivation, the different cell density, the different cell controls used, the different statistical methods used, and finally the differing methodology . Length of incubation periods and differing types of culture (in monolayer, matrix immobilized cultures, or 3d cultures) as well as supplementation of cell culture medium have been shown to have an influence on the composition of the vocs in the samples [79, 81, 8486]. Drug addition also has been shown to change the pattern of vocs produced by a549 cells in vitro, highlighting the possibility of finding biomarkers of apoptosis and necrosis induced by drugs . The main matrices analysed to study vocs generated by cells are (i) hs of the cell - free culture medium of a target cell and (ii) hs of the medium still containing the cells . The hs of cell lysate (preconcentrated supernatant of the lysed cells) is another matrix employed, but has only been used in a few studies, solely for the determination of targeted vocs produced by cancer cells treated with drugs (table 1). There are some substantial differences in terms of the extraction procedure details for the main two matrices . For example, analysis of culture media with cells usually takes place at 37c (physiological conditions), while analysis of media only may employ a higher temperature . Also, the efficiency of analysis of media only samples can be improved by the addition of salts or by a change of ph, while such changes are not possible when cells are present . On the other hand, the analysis of media with cells ensures that no vocs are lost during storage . Some researchers use glass vials as they have very limited release of volatile chemicals (other materials such as standard plastic flasks for cell culture release plasticizers generating additional peaks) [69, 88]. A recent review by kalluri et al . Makes a case that the studies in vivo and in vitro, investigating vocs as potential biomarkers of cancer, have poor correlations (specifically studies of lung cancer and exhaled breath as a sample matrix). They postulate that the overlap between vocs found in the exhaled breath of lung cancer patients and compounds produced by lung cancer cells in vitro (approximately one - quarter being common to both matrices) is not sufficient at the moment for in vitro culture to be a good model for the vocs present in exhaled breath . The authors propose that it could be due to cell cultivation in hyperoxic conditions (atmospheric oxygen concentration) emphasising it as a potential limitation of the in vitro studies performed to date . Tumours have been shown to grow in hypoxic (oxygen depleted) or anoxic (oxygen absent) conditions as opposed to normal tissues . Cellular oxidative stress would lead to the production of different vocs by cells in comparison to hyperoxic cell culture conditions . Studies comparing the patterns of vocs present in the hs of cells cultured in hyperoxic and hypoxic conditions are needed to address this potential limitation of in vitro approach . However, another issue related to cell culture conditions could also result in the different vocs present in the hs of cell culture and samples taken from the patient . Standard 2d cell culture conditions may have a great impact on the cell metabolic behaviour, thereby losing accuracy when looking for biomarkers when compared to 3d culture that better mimics the growth of the tumour . The poor correlation between in vivo and in vitro studies may also arise from exogenous vocs being included in the predictive models of cancer, different extraction and detection techniques used in different studies, different experimental design, and in general a relatively lower number of in vitro studies performed to date, in comparison to the voc studies of breath samples and biofluids . In addition, studies which show that the voc patterns do not change after tumour removal imply that some vocs may be biomarkers of the risk of cancer developing, rather than being indicative of the presence of a tumour (see section 2.4 for further discussion). Also it is important to remember that there is very little known about the complexity of the transmission mechanisms of the vocs produced by tumour cells in the body and found in breath or biofluids . An excellent review of lung cancer voc studies, which describes possible biological pathways of lung cancer vocs identified from different matrices, shows that this is a main challenge to date for cancer voc analysis . Therefore, the composition of vocs found in the samples from the patients and vocs detected in the hs of cultured cells cannot be expected to be the same . However the studies that have been performed to date in order to find potential volatile biomarkers of cancer show that the vocs common to all matrices exist, regardless of the potential limitations of the in vivo and in vitro approaches discussed above . Without doubt, there is a need for a simultaneous investigation of the correlation of the voc pattern in exhaled breath (and other sample types) collected from a patient and an in vitro and/or ex vivo analysis of the vocs produced by the cancer cells or emitted from the cancer tissues (ideally of the same patient). This approach eliminates analytical technique and, in the case of the samples coming from the same patient, factors such as gender, age, and interindividual variation as the sources of possible differences in voc patterns between in vivo, in vitro, and ex vivo samples . Some studies already have been conducted specifically in order to simultaneously compare vocs produced by cancer cells in vitro and ex vivo to the ones found in breath from the patient . The study of chen et al . Aimed to compare vocs produced by four types of primary lung cancer cells to vocs found in cancer breath samples . In this study, 11 vocs were found in breath samples and chosen for principal component analysis in order to discriminate cancer patients from healthy controls, and two compounds were shared with lung cancer cells excised from the patients (namely, isoprene and undecane). Another study compared volatile metabolites determined in a culture medium of lung cancer cell line a549 to the voc composition in the hs of urine of mice implanted with these cells . There were seven vocs found at significantly higher levels in both sample types when compared to normal cancer cell lines (dimethyl succinate, 2-pentanone, phenol, 2-methylpyrazine, 2-hexanone, 2-butanone, and acetophenone). The study performed by buszewski et al . Involved quantitative voc measurement in the hs of healthy and lung cancer tissues and comparison of these results to the ones obtained from the breath samples of the healthy individuals and lung cancer patients . 27 vocs were detected in the air above cancerous tissues, cutting down the number of potential biomarkers that need to be considered when breath samples are analysed . 22 of the same compounds (mainly alcohols, aldehydes, ketones, and aromatic and aliphatic hydrocarbons) were found in the breath samples, just as in the hs of lung tissues . Quantitative analysis of vocs emitted by lung cancer tissues showed higher levels of ethanol, acetone, acetonitrile, 1-propanol, 2-propanol, carbon disulfide, dimethyl sulfide, 2-butanone, and 2-pentanone when compared to control lung tissues . The same compounds were detected in increased concentrations in the breath samples of patients suffering from lung cancer when compared to healthy controls . Some of them were detected in the hs of cancer cells in previous studies (table 2). The exhaled breath of lung cancer patients was compared not only to the breath of healthy controls, but also to the compounds detected in the hs of lung tissues (cancerous and healthy), again in the recent study by filipiak et al . . They detected 39 vocs in both types of samples: tissue specimens, and exhaled breath (with different occurrence ranging from 8 to 100%). Over half of the detected compounds were previously reported in the hs of cancer cells in vitro in different studies (table 2). Although approximately half of the vocs in the breath samples had negative alveolar gradient (alveolar gradient: abundance in breath minus abundance in the air), suggesting their exogenous origin, these findings show common vocs in all three sample types . Out of 39 detected, they found 30 vocs at higher concentrations in cancerous lung tissue, when compared to the healthy tissue controls . Six were elevated at the chosen level of significance: ethanol, pyridine, 4-methylheptane, acetaldehyde, n - octane in the hs of lung cancer tissues, and n - hexanone in the hs of healthy tissues . Ethanol and octane were also found at significantly higher levels in the breath of lung cancer patients . What is more, these compounds were previously detected in the hs of lung cancer cells in vitro . Other vocs found in higher levels in the cancerous lung tissue (but not at significant levels) such as 2-methyl-1-pentene, 4-methyloctane, 2,4-dimethylheptane, hexane, and acetic acid were also previously detected in the hs of different cancer cell lines (table 2). [92, 93] in their unique study measured voc concentrations in the breath of lung cancer patients before and a month and three years after the excision of a tumour . In the study, they analysed 12 vocs that were found in higher concentrations in the breath of cancer patients than in the breath of healthy controls before the surgery . They compared the concentrations of these analytes to the voc levels found in cancerous and healthy lung tissue collected during the surgery . Collection and storage issues allowed for analysis only of aromatic hydrocarbons in the tissue specimens . Six aromatic vocs were found to be common to the exhaled breath and tissue samples (benzene, ethylbenzene, trimethylbenzene, toluene, styrene, and xylenes). Their levels (except for xylenes) were significantly higher in cancerous tissue than in healthy tissue . Ethylbenzene, xylenes, and styrene were compounds detected in the hs of lung cancer cells in previous studies (table 2). Interestingly, no differences in the levels of 11 of the vocs (isoprene being the exception) were found between the breath collected before and one month after the tumour removal . Similar outcomes were obtained by phillips et al . Who did not observe any changes in the voc profiles in the breath of most lung cancer patients before and after surgery . In poli et al . 's study, three years after surgery, the levels of some of these compounds had changed (decreased for isoprene and benzene, increased for pentane, toluene, and ethyl benzene). . 's studies imply that changes in the voc patterns are not biomarkers of lung cancer presence but rather are epiphenomenon of the disease development . 94, 95] proposed an upstream hypothesis which may explain these results as opposed to a downstream hypothesis . In the latter, the presence of the disease causes the altered patterns of vocs in breath samples . In phillips et al . 's pathophysiologic model, altered voc profiles in the breath of the person suffering from lung cancer and the presence of the disease itself are somewhat independent . A person carrying high - risk genotypes due to exposure to carcinogens will have induced activity of the enzymes catabolising vocs . The patterns of the volatile metabolites may therefore be altered before the appearance of the tumour . However, the fact that common vocs were found in the breath, the hs of lung tissues of cancer patients, and the hs of cancer cells grown in vitro, in addition to the fact that the levels of some compounds changed after a longer period following an operation to remove a tumour, implies that at least some of the vocs are produced by the tumour per se and may not be attributable to genetic predispositions . Further studies are required to confirm any of these hypotheses . A different approach to the issue of the possible exogenous origin of proposed voc biomarkers focuses on the detection of aldehydes [12, 59, 65, 96] or hydrocarbons [9799] only as markers of cancer . Studies proved that oxidative stress is one of the main sources of developing cancer via the overproduction of reactive oxygen species and nitrogen species resulting in mutations . Some aldehydes are known to be related to oxidative stress as they are products of lipid peroxidation, but the exact mechanism of their presence in breath and body fluids is not known [27, 101, 102]. This process does not involve branched hydrocarbons as there are no branched polyunsaturated fatty acids in the body, nor does it appear to involve methylated alkanes as there is not enough data to support their origin from lipid peroxidation . As aldehydes are highly reactive and can easily decompose or react while the sample is prepared for analysis or storage, a chemical derivatization has been introduced . One of the most common derivatization methods for aldehyde determination is the reaction of aliphatic aldehydes with pfbha o-(2,3,4,5,6-pentafluorophenyl)methylhydroxylamine hydrochloride to produce stable oximes . Different studies that employed different techniques of extraction demonstrated this as an effective method for aldehyde analysis in various matrices [12, 60, 106108]. Higher concentrations of straight c3c9 aldehydes [32, 65, 96, 106, 108], as well as some unbranched hydrocarbons [28, 93, 97, 99], were identified among vocs in cancerous breath, blood, and urine matrices in many studies . What is more, some of these vocs are the analytes that have been found common to the hs of cancer cells in vitro and exhaled breath of lung cancer patients . Concentrations of most of the vocs present in biological matrices are low: in the nmol pmol (ppb ppt) range in exhaled human breath and in the mol nmol (ppm ppb) range in blood and urine [12, 35, 37, 60]. Therefore, prior to the assay, a preconcentration step is required, which is the most labour - intensive part of the analysis and is the primary source of errors influencing the reliability and accuracy of analysis . Increased reproducibility and elimination of interfering compounds the ideal properties of a sample - preparation device include simplicity, high extraction capacity and selectivity, efficiency, speed, possible automation and miniaturization, compatibility with a range of separation and detection methods, and safety in use for the operator and environment [110, 111]. Microextraction methods employ some of these features the best, when compared to the traditional sampling techniques of liquid - liquid extraction and solid - phase extraction . Solid - phase microextraction (spme) in particular became very popular due to its simplicity and lack of solvent use and the fact that it has been automated and is compatible with gc - ms and lc - ms . Purge and trap (pt) and solid phase microextraction (spme) are the two main extraction techniques used to date for the collection of vocs in both in vivo and in vitro studies of potential cancer biomarkers . In pt (also called dynamic headspace extraction) the gas sample is purged through the sorbent trap by an inert gas and the vocs are retained on the surface of the trap (figure 1). Next they are thermally desorbed with the use of an online thermal desorption (td) device or extracted with small amounts of solvents (liquid desorption: ld). The most commonly used sorbent traps for the analysis of vocs employ charcoal (e.g., carbotrap) or porous polymer (e.g., tenax) as a trapping material with varying degrees of selectivity . Td may cause degradation reactions of sensitive compounds and column degradation, as some sorbents have a high affinity to water . There are various techniques for water removal in pt such as the use of drying gas, a water condenser, or an additional adsorption trap . Ld is a milder technique, so it does not cause degradation of sensitive vocs; however it is less sensitive . In studies of potential voc cancer biomarkers only td - pt has been employed (with cryofocusing to enhance resolution) (e.g., [35, 80]). Spme is an extraction technique where an extraction phase is dispersed on a fine rod made of fused silica, stableflex, or metal alloy . The spme device consists of two parts: the holder and, contained in it, the fiber assembly . There are two versions of the spme holder: one for manual use and one for use with autosamplers or with a high performance liquid chromatography - spme (hplc - spme) interface . The fiber unit consists of a fiber core attached via a hub to a stainless steel guiding rod, which is contained in a hollowed needle that pierces a septum . The fiber is withdrawn from this needle when sampling and the needle is removed when not in use (figure 2). The fiber core is 1 or 2 cm long and is coated with stationary phase . The fiber is immersed in the liquid sample in the case of direct immersion (di - spme) or suspended in the hs above the sample (hs - spme). During extraction, sample molecules preferentially partition from matrix to stationary phase as a result of adsorption or absorption . In the adsorption process the analytes remain on the surface of the trapping material due to chemical bonding . In the absorption process, the analytes are dissolved into the bulk of a liquid phase (e.g., pdms). After sampling, the analytes are thermally desorbed in the injector port with no use of solvents (figure 2). There are several commercially available spme fibers for sampling a wide range of compounds that employ four polymers as stationary phases: divinylbenzene (dvb), polydimethylsiloxane (pdms), polyacrylate (pa), and polyethyleneglycol (peg). They are used on their own as a coat (available in different thicknesses) or in combination blended with carboxen (car). The coatings differ by polarity (polar, bipolar, and nonpolar) and extraction mechanism (absorbent or adsorbent). The choice of fiber coating depends on the polarity of analytes and their molecular weight . Previous analyses of vocs as biomarkers of cancer has been performed in most cases with the use of a 75 m car / pdms coating regardless of the type of matrix tested . Its use is justified, as the fiber was initially developed for the extraction of volatile and small compounds . In comparison, a sorbent trap is an exhaustive extraction technique, due to chemical reactions between the stationary phase and the analytes, whereas spme is a nonexhaustive (passive) equilibrium technique where the amounts of vocs extracted are controlled by the series of distribution constants between the gaseous, liquid, and coating phases . Spme, depending on the fiber used, is an absorption technique or utilizes absorption and adsorption properties simultaneously . Sorbent trapping is a three - step process (extraction of the analytes to the solid sorbent, desorption, and cold focusing), whereas spme is more simple in use (sorption of analytes onto the fiber then desorption). Spme - gc - ms does not require an additional device connected to the gas chromatograph such as a cryotrap or a water removal device . On the other hand, as spme methodology is limited by the commercially available fibres, its sensitivity is also limited . Sorbent traps may employ additional preconcentration, such as using higher volumes of the trapping material to enhance sensitivity . The sensitivity of spme is not as dependent on sample volume as sorbent traps; the limits of detection of the latter technique get better with a larger volume of sample . For example, the use of sorbent traps showed an order of magnitude lower limit of detection (lod) than spme for isoprene in human breath, when the same, 8 l breath samples were analysed . Lods obtained in studies analysing vocs as potential cancer biomarkers showed that pt extraction technique yielded better sensitivity (low ngl in full scan mode) than spme (gl in full scan mode) [80, 119]. As selection of the appropriate fiber coating for analytes is a critical stage in the spme methodology development, there are new fiber coatings under development with higher capacity and selectivity, which would enhance sensitivity such as molecularly imprinted polymers, multiwall carbon nanotubes, sol - gel technology, and polymeric ionic liquids (reviewed in [120, 121]). Other variations of spme techniques such as stir bar sorptive extraction (sbse), solid phase microextraction membrane, (or thin - film microextraction), and needle trap device have been successfully used for the collection of vocs and so may be used in cancer studies in the future (reviewed in [111, 120]). Needle trap device has been already used by mochalski et al . For analysis of vocs in the hs of liver cancer cell line . Another microextraction method, single drop microextraction, was also introduced for the hs analysis of vocs in cancerous blood . The technique is simple and rapid, uses trace amounts of solvents (2 l), and is less costly than spme . [73, 123] used ultra ii skc passive (no purge) diffusion badges for the preconcentration of vocs from the hs of the cell culture media . In this type of sampler sorbent traps serve as adsorption material, and extraction is based, as in spme, on the equilibrium principles . (with the use of td). Finally, cryoconcentration was also used prior to analysis in a study, in order to investigate vocs produced by leukaemia cell line . The main detection techniques that have been employed in voc cancer biomarker studies are gc - ms, proton transfer reaction - mass spectrometry (ptr - ms), selected ion flow tube - mass spectrometry (sift - ms), and gas sensors (electronic noses) (table 3). Sampling and analytical techniques for the analysis of vocs in biological samples are summarised in the recent review by zhang et al . And for breath analysis specifically in the reviews by di francesco et al . Advances and/or applications in gas sensor technology in breath analysis have been recently described in a number of reviews [126132]. Gc - ms is the most commonly used analytical technique for the investigation of potential voc cancer biomarkers, due to its sensitivity and reliability in analyte identification . It gives the most detailed analytical information and identifies analytes with the most certainty, when compared to ptr - ms . The identification of vocs with the use of ptr - ms can be tentative only as it is not possible to discriminate between compounds with the same molecular weight [74, 88, 133]. On the other hand, ptr - ms is the most sensitive method of all, with the limit of detection for aromatic hydrocarbons in low - ppb levels, or even as low as a few ppt . It has been demonstrated to be more sensitive than gc - ms measurement by a factor of ~20 . Gc - ms was shown to have sensitivity for voc analysis at the ppb and low ppt levels but it needs a further preconcentration step [96, 117]. Sift - ms allows for the measurement of trace gases at sub - ppb levels, but it is also reliable in the identification of compounds . The advantage of ptr - ms and sift - ms over gc - ms is that they do not require a preconcentration step and can work in online (real time) mode . Therefore they are better techniques for the quantification of vocs, as they provide instant quantification of all the analytes in the sample [133, 136] (table 3). In comparison, spme - gc - ms measures analytes semiquantitatively, as it involves competitive absorption of the compounds on the fiber . Nevertheless, instruments for all the techniques are not easy to use in clinical settings in terms of portability or transport . Although the easily transportable sift (transsift) and ptr (ptr - qms 300) instruments have been introduced commercially [136, 137], their small sizes compromise their sensitivity . Another detection technique, ion mobility spectrometry (ims), is not very common yet in the studies of vocs as potential cancer biomarkers, but already has shown promising results . The first study which applied ims for the analysis of vocs in the exhaled breath of lung cancer patients and healthy subjects discriminant analysis employing 23 voc peaks identified individuals with or without a tumour with 100% accuracy . In another study, the detection of different voc concentrations in the breath of cancer patients using ims allowed for discrimination between different histological subtypes of lung cancer . The ims detector is characterised by low selectivity . Therefore, complex mixtures are analysed with the use of a preseparation technique such as multicapillary column (mcc) or gc [140, 141]. Mainly ims coupled with mcc has been used for breath analysis in the studies performed to date [138140, 142]. The advantages of mcc - ims include very fast analysis (500 s for the breath sample), no need for preconcentration, and online analysis . In contrast to other analytical techniques, the use of mcc - ims allows for the detection of all the analytes in a breath sample through their separation by retention time, mobility, and concentration and by creating a 3d visualisation of each compound in the chromatogram . Although it does not allow for the identification of the analytes, ims is a comparatively cheap detection technique with a potential for miniaturisation and is therefore one of the most promising, next to electronic noses, candidates to be used in a clinical setting . Compared to mass spectrometric methods, the use of electronic noses does not require skilled personnel and is less time consuming . These features, as well as the potential miniaturisation of such devices, make them ideal potential diagnostic tools to be used by general practitioners or even as devices for personal use . There have been several types of electronic noses used in the studies of vocs in cancer [33, 73, 143, 145147]. They are designed to recognise voc patterns emitted from the analysed samples, but not to identify these vocs . Quantification of vocs with the use of an electronic nose has not been performed in any studies of cancer . In terms of breath testing, such sensor systems could be cheap, rapid, and simple to use when they have been tailored for a specific use . However, electronic noses are highly sensitive to moisture and relatively less sensitive (15 ppb) and their effectiveness needs more validation studies as they have shown poor linearity and reproducibility . Nevertheless, electronic noses constitute a very promising research area in the analysis of vocs as potential cancer biomarkers . For example, a novel combination of a gc separation system and metal oxide sensor device has already shown very good accuracy in diagnosing bladder cancer . Quartz microbalance gas sensors also demonstrated very good accuracy in differentiation between lung cancer patients and healthy controls . Finally nanomaterial - based chemiresistors have shown the ability to distinguish not only between the breath of patients suffering from different cancer types, but also between early and advanced stages of lung cancer, between the types of lung cancer, and between malignant and benign pulmonary nodules . The discussion starts with the issue of whether to choose an in vivo or in vitro system for study . Obviously the aim is to apply differential vocs of cancer to a device that will enable the detection of cancer in the patient with 100% certainty, ideally noninvasively, as the less invasive a procedure is, the cheaper and more simple it will be to conduct . Whether it is going to be breath, blood, urine, or any other sample coming from the patient, at this stage, none of these matrices is ideal for looking for potential volatile biomarkers . The main reason is the uncertain origin of the detected vocs, as their patterns may depend not only on the presence of the disease, but also on the long list of other variables such as genetic and environmental factors, age, gender, and so forth . Therefore, it seems obvious to complement these studies with an investigation of the voc profiles produced by tumours at the microcellular level, where an explanation of the presence of a compound in the chromatogram is more straightforward . The studies on cells are of great informative value about the biochemistry of tumours . However, with the in vitro approach, there are also some uncertainties arising . The main one is that there is little known about the complexity of vocs metabolic pathways, between the voc being produced by the tumour cells and its presence (or absence) in the sample from the patient . Nevertheless, in vitro studies are valuable tools in advancing the aim of cancer diagnosis . Ideally, research should be directed to comparing voc patterns in the hs of cancer cells or tissues of one particular patient with the compounds detected in breath, urine, and/or blood of the same patient . Also the selection of controls is crucial, in order to eliminate as many variables as possible . Without doubt, more studies are needed for the comparison of vocs produced by tumour cells to the ones found in breath or biofluids, as well as for comparison of voc patterns generated by many cell lines and primary tumour samples in order to profile as many cells as possible, so that an attempt can be made to find the common vocs for particular types of cancer . Each of the five types of analytical techniques that found application in the studies of vocs has its advantages and disadvantages . Although it is more likely that a future tool to be used in the clinic will be an electronic sensor device, due to its cheaper cost, however, gas sensors still have poor sensitivities . Therefore other analytical techniques may be researched further . Consequently, for research purposes, it seems to be ideal to use the methods in complement . Studies of the scent of cancer are really elegant in the simplicity of the idea; however there are still limitations of applying this idea clinically regardless of the technique used . At the moment certainty analysis of breath and other matrices investigating potential biomarkers of cancer is still in its infancy . Evidently large - scale screening studies are first required in order to describe normal profiles of vocs in all matrices being studied . Knowledge about voc concentration ranges for a normal, nondiseased state and validation studies using larger populations in relation to all forms of cancer will further evaluate the promising results of the existing studies of these diseases . And here surely the path to the use of vocs as smellprints of cancer in the clinic lies in using information gleaned from a variety of different approaches in complement.
To explore the experiences of patients with cancer who were offered a nurse navigator in their course of illness before the in - hospital period . Development has fragmentized healthcare systems in many countries, and coherence is now desired . Among interventions suggested to reduce the fragmentation and improve delivery of care are help from patient navigators, where patients are offered extra help in a defined area by e.g., a nurse [nurse navigator (nn)]. A phenomenological - hermeneutical longitudinal study was performed among danish gynecological patients from before an in - hospital period to two months after discharge . Not all could use the help from nn . Those who could, attached affectional bonds to nn and experienced benefit from her presence as well as her help . Many had a feeling of deep - felt disappointment and felt rejected when the contact to nn stopped . Resources for nn should be prioritized to patients who can use the help, and not stop prematurely . The traditional division and thinking by healthcare professionals are challenged, if all patients should be helped.
Patients with neuropathic pain experience spontaneous tingling sensations, hyperalgesia, and tactile allodynia, symptoms that deteriorate their quality of life . The pathogenesis of neuropathic pain is believed to involve central sensitization of spinal dorsal horn neurons following central or peripheral nervous system damage . Glia interactions play a crucial role in the development of central sensitization . Various cytokines or chemokines interferon - gamma (ifn) is a pro - inflammatory cytokine and a major modulator of central and peripheral immune responses that plays roles in a variety of chronic pain conditions . For example, patients with persistent pain due to viral infections or multiple sclerosis have persistently high ifn levels in the spinal cord as an inflammatory response . This cytokine is produced primarily by t - cells and natural killer cells and stimulates the janus activated kinase (jak)-signal transducer and activator of transcription (stat) signaling pathway . Upon peripheral nerve injury, ifn is upregulated in the rat dorsal horn as t - cells that infiltrate the spinal cord, and it has been implicated as one of the causative agents of neuropathic pain . Interestingly, intrathecal injections of ifn induce pain - related behaviors in wildtype rats and mice but not ifn receptor knock - out mice . However, little is known about the roles of ifn in the dorsal horn of the spinal cord . The substantia gelatinosa (sg, lamina ii) of the spinal cord dorsal horn has been implicated in nociceptive transmission because both finely myelinated (a) and unmyelinated (c) primary nociceptive afferent fibers are heavily projected to this spinal region . To elucidate how ifn affects nociceptive responses in the spinal dorsal horn, we analyzed the effects of ifn on postsynaptic action by using whole - cell patch - clamp recordings from sg neurons . Five to six - week - old male sprague dawley rats (180210 g; obtained from slc, japan) were used . The protocol was approved by the ethics committee on animal experiments, wakayama medical university, and was in accordance with the uk animals (scientific procedures) act of 1986 and associated guidelines . Animals were housed in plastic cages at room temperature on a 12-h light / dark cycle (light on between 8:00 a.m. and 8:00 p.m.) with ad libitum access to food and water . Briefly, the adult rats were anesthetized with urethane (1.2 g / kg, intraperitoneal), and th10-l4 laminectomy was performed . Then, we excised the spinal cord and submerged it in preoxygenated krebs solution at 13. immediately after the removal of the spinal cord, the rats were killed by exsanguination under urethane . The dura, arachnoid, and pia mater were removed, and the spinal segment was sliced to 650-m thickness by using a microslicer (dtk-1000, dousaka from kyoto, japan). The slices were continuously perfused with krebs solution (10 ml / min) and saturated with 95% o2 and 5% co2 . The perfusion solution was heated to 36 1 by using a temperature controller . The krebs solution contained the following (in mm): 117 nacl, 3.6 kcl, 1.2 nah2po4, 2.5 cacl2, 1.2 mgcl2, 25 nahco3, and 11 glucose, at ph 7.4 . Blind whole - cell patch - clamp recordings were made from lamina ii (sg) neurons in voltage - clamp mode . The patch - pipette had a resistance of 510, and the composition of the patch - pipette internal solution was as follows (in mm): 135 potassium gluconate, 5 kcl, 0.5 cacl2, 2 mgcl2, 5 egta, 5 hepes, and 5 atp mg . Membrane currents were amplified by using an amplifier (axopatch 200b from molecular devices, sunnyvale, ca, usa) in voltage - clamp mode . Holding potential was set to 70 mv for recording exogenous -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (ampa) current and to 50 mv for n - methyl - d - aspartate (nmda) current . Data were digitized using an analog - to - digital converter (digidata 1440a from molecular devices), stored, and then analyzed using a personal computer with the pclamp 10 data acquisition program (molecular devices). The drugs used in this study were ifn (peprotech, rocky hill, nj, usa); ifn antagonist (vwr international, radnor, pa, usa); monocyte chemoattractant protein 1 (mcp-1) (r&d, minneapolis, mn, usa); and, ampa, nmda, minocycline hydrochloride, teijin compound 1 hydrochloride, gdp--s, and tofacitinib citrate (sigma aldrich, st . Ifn antagonist, mcp-1, ampa, nmda, and minocycline hydrochloride were dissolved in distilled water as 1,000 stock solutions, and teijin compound 1 hydrochloride and tofacitinib citrate were first dissolved in 5% dmso as 1,000 stock solutions . The 1,000 stock solutions of ifn were prepared in distilled water containing 1% bovine serum albumin (bsa) (sigma aldrich). All perfusion drugs were stored at 20 and diluted to the final concentration in krebs solution just before use, and then were perfused via a three - way stopcock without any change in the rate or temperature . The time necessary for the solution to flow from the stopcock to the spinal cord section surface was about 1 min . The tubing of our perfusion system was coated with aquasil siliconizing fluid (thermo fisher scientific, waltham, ma, usa) to reduce the loss of ifn through nonspecific interactions . All numerical data are expressed as mean standard error (se). Paired student s t tests were used for statistical comparison, and the criterion for statistical significance was p <0.05; n refers to the number of neurons studied . Briefly, the adult rats were anesthetized with urethane (1.2 g / kg, intraperitoneal), and th10-l4 laminectomy was performed . Then, we excised the spinal cord and submerged it in preoxygenated krebs solution at 13. immediately after the removal of the spinal cord, the rats were killed by exsanguination under urethane . The dura, arachnoid, and pia mater were removed, and the spinal segment was sliced to 650-m thickness by using a microslicer (dtk-1000, dousaka from kyoto, japan). The slices were continuously perfused with krebs solution (10 ml / min) and saturated with 95% o2 and 5% co2 . The perfusion solution was heated to 36 1 by using a temperature controller . The krebs solution contained the following (in mm): 117 nacl, 3.6 kcl, 1.2 nah2po4, 2.5 cacl2, 1.2 mgcl2, 25 nahco3, and 11 glucose, at ph 7.4 . Blind whole - cell patch - clamp recordings were made from lamina ii (sg) neurons in voltage - clamp mode . The patch - pipette had a resistance of 510, and the composition of the patch - pipette internal solution was as follows (in mm): 135 potassium gluconate, 5 kcl, 0.5 cacl2, 2 mgcl2, 5 egta, 5 hepes, and 5 atp mg . Membrane currents were amplified by using an amplifier (axopatch 200b from molecular devices, sunnyvale, ca, usa) in voltage - clamp mode . Holding potential was set to 70 mv for recording exogenous -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (ampa) current and to 50 mv for n - methyl - d - aspartate (nmda) current . Data were digitized using an analog - to - digital converter (digidata 1440a from molecular devices), stored, and then analyzed using a personal computer with the pclamp 10 data acquisition program (molecular devices). The drugs used in this study were ifn (peprotech, rocky hill, nj, usa); ifn antagonist (vwr international, radnor, pa, usa); monocyte chemoattractant protein 1 (mcp-1) (r&d, minneapolis, mn, usa); and, ampa, nmda, minocycline hydrochloride, teijin compound 1 hydrochloride, gdp--s, and tofacitinib citrate (sigma aldrich, st . Ifn antagonist, mcp-1, ampa, nmda, and minocycline hydrochloride were dissolved in distilled water as 1,000 stock solutions, and teijin compound 1 hydrochloride and tofacitinib citrate were first dissolved in 5% dmso as 1,000 stock solutions . The 1,000 stock solutions of ifn were prepared in distilled water containing 1% bovine serum albumin (bsa) (sigma aldrich). All perfusion drugs were stored at 20 and diluted to the final concentration in krebs solution just before use, and then were perfused via a three - way stopcock without any change in the rate or temperature . The time necessary for the solution to flow from the stopcock to the spinal cord section surface was about 1 min . The tubing of our perfusion system was coated with aquasil siliconizing fluid (thermo fisher scientific, waltham, ma, usa) to reduce the loss of ifn through nonspecific interactions . All numerical data are expressed as mean standard error (se). Paired student s t tests were used for statistical comparison, and the criterion for statistical significance was p <0.05; n refers to the number of neurons studied . Only sg neurons, with resting membrane potentials lower than 50 mv in the current - clamp mode, were used for recording . Perfusion of exogenous ampa (10 m) for 30 s onto the spinal cord slice induces slow inward currents at a holding potential of 70 mv, and nmda (50 m) does so at 50 mv . This discrepancy is because nmda receptors are blocked by mg when the cell membrane is held at 70 mv . Ampa superfusion repeatedly produces an inward current with the same peak amplitude in an sg neuron (101.3 0.6%, n = 5, p = 0.09), observed for nmda (97.9 3.1%, we first examined the effects of ifn on inward currents induced by ampa and nmda when maintaining the voltage at 70 and 50 mv, respectively . Bath application of ifn (30 nm, 2.5 min) did not affect the ampa - induced current (100.0 3.7%, n = 11, p = 0.87, mean amplitude of control: 51.5 pa; figure 1(a),(c)). On the other hand, nmda - induced current was significantly increased by ifn (147.3 10.7%, n = 14, p = 0.0002, mean amplitude of control: 16.8 pa; figure 1(b),(d)). This ifn-induced facilitation of nmda current was inhibited by an ifn receptor - selective antagonist (300 nm, 5.5 min) (90.0 3.6%, n = 10, p = 0.053; figure 2). These results suggest that ifn binds to own receptor and enhances the activity of nmda but not ampa receptors in the dorsal horn of the spinal cord . Figure 1.ampa- and nmda - induced currents under bath application of ifn in adult rat sg neurons . (a) recordings of ampa - induced inward currents at a holding potential of 70 mv . Perfusion of ifn (30 nm, 2.5 min) did not result in any significant change in ampa - induced currents . (b) effects of ifn on nmda - induced inward currents at a holding potential of 50 mv . Nmda - induced current was significantly increased by ifn (30 nm, 2.5 min). (c) the peak amplitude of the ampa - induced currents after ifn treatment was 100.0 3.7% of baseline . (d) the peak amplitude of the nmda - induced currents after ifn treatment was 147.3 10.7% of baseline . In figure 1(b) and subsequent figures, nmda responses were recorded in voltage - clamp mode (vh = 50 mv). N.s . : not significant . * ampa: -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid; ifn: interferon - gamma; nmda: n - methyl - d - aspartate . (a) perfusion of the ifn receptor - selective antagonist (300 nm, 5.5 min) inhibited the ifn-induced facilitation of nmda current . (b) bar graphs show the peak amplitude of nmda - induced current compared to baseline when the ifn receptor - selective antagonist and ifn are perfused simultaneously . Therefore, ifn likely binds to ifn receptors and activates nmda receptors in sg neurons . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate . Ampa- and (a) recordings of ampa - induced inward currents at a holding potential of 70 mv . Perfusion of ifn (30 nm, 2.5 min) did not result in any significant change in ampa - induced currents . (b) effects of ifn on nmda - induced inward currents at a holding potential of 50 mv . Nmda - induced current was significantly increased by ifn (30 nm, 2.5 min). (c) the peak amplitude of the ampa - induced currents after ifn treatment was 100.0 3.7% of baseline . (d) the peak amplitude of the nmda - induced currents after ifn treatment was 147.3 10.7% of baseline . In figure 1(b) and subsequent figures, nmda responses were recorded in voltage - clamp mode (vh = 50 mv). N.s . : not significant . * p <0.05 . Ampa: -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid; ifn: interferon - gamma; nmda: n - methyl - d - aspartate . (a) perfusion of the ifn receptor - selective antagonist (300 nm, 5.5 min) inhibited the ifn-induced facilitation of nmda current . (b) bar graphs show the peak amplitude of nmda - induced current compared to baseline when the ifn receptor - selective antagonist and ifn are perfused simultaneously . Therefore, ifn likely binds to ifn receptors and activates nmda receptors in sg neurons . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate . Because both neurons and microglia are reported as sites of ifn receptors, we investigated as shown in figure 3, the ifn-induced facilitation of nmda currents was inhibited by minocycline (20 m, 5.5 min), an inhibitor of microglia activation (90.3 6.4%, n = 12, p = 0.12). In addition, we recorded the ifn-induced facilitation of nmda current during inhibition of jak - stat signaling . We added the jak inhibitor tofacitinib (1 mm) to the pipette solution . Despite blocking jak - stat signaling, ifn-induced facilitation of nmda currents was not affected even after 30 min (98.9 6.0%, n = 7, p = 0.99; figure 4). Therefore, we considered that ifn activates the microglial ifn receptor and enhances the activity of nmda receptors in the dorsal horn neuron . (a) minocycline (20 m, 5.5 min), an inhibitor of microglia activation, inhibited the ifn-induced facilitation of the nmda current . (b) bar graphs show the peak amplitude of the nmda - induced currents compared with baseline when minocycline and ifn were perfused simultaneously . Therefore, ifn likely binds to microglial ifn receptors and enhances the activity of nmda receptors in dorsal horn neurons . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate . (a) tofacitinib (1 mm) addition to the pipette solution did not affect the ifn-induced nmda current . In this experimental system, the control corresponds to the ifn-induced nmda currents recorded immediately after patch clamping . (b) bar graphs show the average rate of nmda current enhancement by ifn at the start of patch - clamp recording and 30 min later . The peak amplitude of the ifn-induced nmda currents did not change compared with the control recording . This confirmed that ifn enhances neuronal nmda - induced inward current via the microglial ifn receptor . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate; n.s . : (a) minocycline (20 m, 5.5 min), an inhibitor of microglia activation, inhibited the ifn-induced facilitation of the nmda current . (b) bar graphs show the peak amplitude of the nmda - induced currents compared with baseline when minocycline and ifn were perfused simultaneously . Therefore, ifn likely binds to microglial ifn receptors and enhances the activity of nmda receptors in dorsal horn neurons . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate . (a) tofacitinib (1 mm) addition to the pipette solution did not affect the ifn-induced nmda current . In this experimental system (b) bar graphs show the average rate of nmda current enhancement by ifn at the start of patch - clamp recording and 30 min later . The peak amplitude of the ifn-induced nmda currents did not change compared with the control recording . This confirmed that ifn enhances neuronal nmda - induced inward current via the microglial ifn receptor . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate; n.s . : not significant . As jak - stat signaling was not the intraneuronal signal transducer underlying the effect of ifn on nmda currents, we examined another possible signaling pathway . First, we added 1 mm gdp--s, a non - hydrolysable analog of gdp that competitively inhibits g proteins, to the pipette solution . Nmda - induced inward currents were significantly suppressed when ifn and nmda were applied 30 min after patch clamping with pipettes containing gdp--s (78.7 5.1%, n = 6, p = 0.023; figure 5). These findings suggested that the ifn-induced increase of nmda currents involves the activation of g protein - coupled receptors such as chemokine receptors . Specifically, the chemokine mcp-1, also known as chemokine ligand 2, ccl2 and its receptor c - c chemokine receptor type 2 (ccr2) are reportedly involved in the generation of neuropathic pain and are related to ifn. We then investigated the participation of ccl2/ccr2 in this effect of ifn on nmda currents . Ifn-induced enhancement of nmda currents was blocked by a selective antagonist of ccr2, teijin compound 1 hydrochloride (10 m, 5.5 min) (95.0 5.3%, n = 6, p = 0.43; figure 6). In addition, bath - applied ccl2 (100 ng / ml) for 2.5 min enhanced nmda - induced inward current (128.3 6.4%, n, it is possible that microglia activated by ifn release ccl2, which binds to ccr2 and enhances neuronal nmda - induced current . (a) in a potassium gluconate pipette solution containing gdp--s (1 mm), the ifn-induced nmda current after 30 min of recording was significantly lower than the control recording of ifn-induced nmda currents recorded at the start of patch clamping . (b) bar graphs show the average rate of ifn-induced enhancement of nmda current in the same neuron at the beginning of the recording and after 30 min . The value of the right bar is 78.7 5.1% of the left bar . This finding suggested that the ifn-induced enhancement of nmda currents involves the activation of g protein - coupled receptors . * p <0.05 . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate . Figure 6.the enhancement of nmda - induced inward current by ifn was mediated by ccr2 . (a) bath application of the ccr2 antagonist teijin compound 1 hydrochloride (10 m, 5.5 min) blocked the ifn-induced enhancement of nmda currents . (b) the peak amplitude of the nmda - induced currents after simultaneous application of teijin compound 1 hydrochloride and ifn was 95.0 5.3% that of baseline . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate; n.s . (a) perfusion of spinal cord slices with ccl2 (100 ng / ml, 2.5 min) increased nmda - induced inward currents . (b) the peak amplitude of the nmda - induced currents after simultaneous ccl2 application is 128.3 6.4% compared with baseline . This suggests that ifn increases the nmda - induced inward current in sg neurons via ccl2/ccr2 signaling . * mcp-1: monocyte chemoattractant protein 1; nmda: n - methyl - d - aspartate . (a) in a potassium gluconate pipette solution containing gdp--s (1 mm), the ifn-induced nmda current after 30 min of recording was significantly lower than the control recording of ifn-induced nmda currents recorded at the start of patch clamping . (b) bar graphs show the average rate of ifn-induced enhancement of nmda current in the same neuron at the beginning of the recording and after 30 min . The value of the right bar is 78.7 5.1% of the left bar . This finding suggested that the ifn-induced enhancement of nmda currents involves the activation of g protein - coupled receptors . * p <0.05 . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate . (a) bath application of the ccr2 antagonist teijin compound 1 hydrochloride (10 m, 5.5 min) blocked the ifn-induced enhancement of nmda currents . (b) the peak amplitude of the nmda - induced currents after simultaneous application of teijin compound 1 hydrochloride and ifn was 95.0 5.3% that of baseline . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate; n.s . (a) perfusion of spinal cord slices with ccl2 (100 ng / ml, 2.5 min) increased nmda - induced inward currents . (b) the peak amplitude of the nmda - induced currents after simultaneous ccl2 application is 128.3 6.4% compared with baseline . This suggests that ifn increases the nmda - induced inward current in sg neurons via ccl2/ccr2 signaling . * mcp-1: monocyte chemoattractant protein 1; nmda: n - methyl - d - aspartate . Only sg neurons, with resting membrane potentials lower than 50 mv in the current - clamp mode, were used for recording . Perfusion of exogenous ampa (10 m) for 30 s onto the spinal cord slice induces slow inward currents at a holding potential of 70 mv, and nmda (50 m) does so at 50 mv . This discrepancy is because nmda receptors are blocked by mg when the cell membrane is held at 70 mv . Ampa superfusion repeatedly produces an inward current with the same peak amplitude in an sg neuron (101.3 0.6%, n = 5, p = 0.09), observed for nmda (97.9 3.1%, we first examined the effects of ifn on inward currents induced by ampa and nmda when maintaining the voltage at 70 and 50 mv, respectively . Bath application of ifn (30 nm, 2.5 min) did not affect the ampa - induced current (100.0 3.7%, n = 11, p = 0.87, mean amplitude of control: 51.5 pa; figure 1(a),(c)). On the other hand, nmda - induced current was significantly increased by ifn (147.3 10.7%, n = 14, p = 0.0002, mean amplitude of control: 16.8 pa; figure 1(b),(d)). This ifn-induced facilitation of nmda current was inhibited by an ifn receptor - selective antagonist (300 nm, 5.5 min) (90.0 3.6%, n = 10, p = 0.053; figure 2). These results suggest that ifn binds to own receptor and enhances the activity of nmda but not ampa receptors in the dorsal horn of the spinal cord . Figure 1.ampa- and nmda - induced currents under bath application of ifn in adult rat sg neurons . (a) recordings of ampa - induced inward currents at a holding potential of 70 mv . Perfusion of ifn (30 nm, 2.5 min) did not result in any significant change in ampa - induced currents . (b) effects of ifn on nmda - induced inward currents at a holding potential of 50 mv . Nmda - induced current was significantly increased by ifn (30 nm, 2.5 min). (c) the peak amplitude of the ampa - induced currents after ifn treatment was 100.0 3.7% of baseline . (d) the peak amplitude of the nmda - induced currents after ifn treatment was 147.3 10.7% of baseline . In figure 1(b) and subsequent figures, nmda responses were recorded in voltage - clamp mode (vh = 50 mv). N.s . : not significant . * ampa: -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid; ifn: interferon - gamma; nmda: n - methyl - d - aspartate . (a) perfusion of the ifn receptor - selective antagonist (300 nm, 5.5 min) inhibited the ifn-induced facilitation of nmda current . (b) bar graphs show the peak amplitude of nmda - induced current compared to baseline when the ifn receptor - selective antagonist and ifn are perfused simultaneously . Therefore, ifn likely binds to ifn receptors and activates nmda receptors in sg neurons . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate . Ampa- and (a) recordings of ampa - induced inward currents at a holding potential of 70 mv . Perfusion of ifn (30 nm, 2.5 min) did not result in any significant change in ampa - induced currents . (b) effects of ifn on nmda - induced inward currents at a holding potential of 50 mv . Nmda - induced current was significantly increased by ifn (30 nm, 2.5 min). (c) the peak amplitude of the ampa - induced currents after ifn treatment was 100.0 3.7% of baseline . (d) the peak amplitude of the nmda - induced currents after ifn treatment was 147.3 10.7% of baseline . In figure 1(b) and subsequent figures, nmda responses were recorded in voltage - clamp mode (vh = 50 mv). N.s . : not significant . * p <0.05 . Ampa: -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid; ifn: interferon - gamma; nmda: n - methyl - d - aspartate . (a) perfusion of the ifn receptor - selective antagonist (300 nm, 5.5 min) inhibited the ifn-induced facilitation of nmda current . (b) bar graphs show the peak amplitude of nmda - induced current compared to baseline when the ifn receptor - selective antagonist and ifn are perfused simultaneously . Therefore, ifn likely binds to ifn receptors and activates nmda receptors in sg neurons . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate . Because both neurons and microglia are reported as sites of ifn receptors, we investigated which receptor is responsible for the ifn-induced potentiation of nmda - induced currents . As shown in figure 3, the ifn-induced facilitation of nmda currents was inhibited by minocycline (20 m, 5.5 min), an inhibitor of microglia activation (90.3 6.4%, n = 12, p = 0.12). In addition, we recorded the ifn-induced facilitation of nmda current during inhibition of jak - stat signaling . We added the jak inhibitor tofacitinib (1 mm) to the pipette solution . Despite blocking jak - stat signaling, ifn-induced facilitation of nmda currents was not affected even after 30 min (98.9 6.0%, n = 7, p = 0.99; figure 4). Therefore, we considered that ifn activates the microglial ifn receptor and enhances the activity of nmda receptors in the dorsal horn neuron . (a) minocycline (20 m, 5.5 min), an inhibitor of microglia activation, inhibited the ifn-induced facilitation of the nmda current . (b) bar graphs show the peak amplitude of the nmda - induced currents compared with baseline when minocycline and ifn were perfused simultaneously . Therefore, ifn likely binds to microglial ifn receptors and enhances the activity of nmda receptors in dorsal horn neurons . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate . (a) tofacitinib (1 mm) addition to the pipette solution did not affect the ifn-induced nmda current . In this experimental system (b) bar graphs show the average rate of nmda current enhancement by ifn at the start of patch - clamp recording and 30 min later . The peak amplitude of the ifn-induced nmda currents did not change compared with the control recording . This confirmed that ifn enhances neuronal nmda - induced inward current via the microglial ifn receptor . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate; n.s . : not significant . (a) minocycline (20 m, 5.5 min), an inhibitor of microglia activation, inhibited the ifn-induced facilitation of the nmda current . (b) bar graphs show the peak amplitude of the nmda - induced currents compared with baseline when minocycline and ifn were perfused simultaneously . Therefore, ifn likely binds to microglial ifn receptors and enhances the activity of nmda receptors in dorsal horn neurons . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate . (a) tofacitinib (1 mm) addition to the pipette solution did not affect the ifn-induced nmda current . In this experimental system, the control corresponds to the ifn-induced nmda currents recorded immediately after patch clamping . (b) bar graphs show the average rate of nmda current enhancement by ifn at the start of patch - clamp recording and 30 min later . The peak amplitude of the ifn-induced nmda currents did not change compared with the control recording . This confirmed that ifn enhances neuronal nmda - induced inward current via the microglial ifn receptor . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate; n.s . : not significant . As jak - stat signaling was not the intraneuronal signal transducer underlying the effect of ifn on nmda currents, we examined another possible signaling pathway . First, we added 1 mm gdp--s, a non - hydrolysable analog of gdp that competitively inhibits g proteins, to the pipette solution . Nmda - induced inward currents were significantly suppressed when ifn and nmda were applied 30 min after patch clamping with pipettes containing gdp--s (78.7 5.1%, n = 6, p = 0.023; figure 5). These findings suggested that the ifn-induced increase of nmda currents involves the activation of g protein - coupled receptors such as chemokine receptors . Specifically, the chemokine mcp-1, also known as chemokine ligand 2, ccl2 and its receptor c - c chemokine receptor type 2 (ccr2) are reportedly involved in the generation of neuropathic pain and are related to ifn. We then investigated the participation of ccl2/ccr2 in this effect of ifn on nmda currents . Ifn-induced enhancement of nmda currents was blocked by a selective antagonist of ccr2, teijin compound 1 hydrochloride (10 m, 5.5 min) (95.0 5.3%, n = 6, p = 0.43; figure 6). In addition, bath - applied ccl2 (100 ng / ml) for 2.5 min enhanced nmda - induced inward current (128.3 6.4%, n, it is possible that microglia activated by ifn release ccl2, which binds to ccr2 and enhances neuronal nmda - induced current . Figure 5.the g protein inhibitor gdp--s significantly inhibited ifn-induced nmda currents . (a) in a potassium gluconate pipette solution containing gdp--s (1 mm), the ifn-induced nmda current after 30 min of recording was significantly lower than the control recording of ifn-induced nmda currents recorded at the start of patch clamping . (b) bar graphs show the average rate of ifn-induced enhancement of nmda current in the same neuron at the beginning of the recording and after 30 min . The value of the right bar is 78.7 5.1% of the left bar . This finding suggested that the ifn-induced enhancement of nmda currents involves the activation of g protein - coupled receptors . * p <0.05 . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate . Figure 6.the enhancement of nmda - induced inward current by ifn was mediated by ccr2 . (a) bath application of the ccr2 antagonist teijin compound 1 hydrochloride (10 m, 5.5 min) blocked the ifn-induced enhancement of nmda currents . (b) the peak amplitude of the nmda - induced currents after simultaneous application of teijin compound 1 hydrochloride and ifn was 95.0 5.3% that of baseline . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate; n.s . : not significant . (a) perfusion of spinal cord slices with ccl2 (100 ng / ml, 2.5 min) increased nmda - induced inward currents . (b) the peak amplitude of the nmda - induced currents after simultaneous ccl2 application is 128.3 6.4% compared with baseline . This suggests that ifn increases the nmda - induced inward current in sg neurons via ccl2/ccr2 signaling . Mcp-1: monocyte chemoattractant protein 1; nmda: n - methyl - d - aspartate . (a) in a potassium gluconate pipette solution containing gdp--s (1 mm), the ifn-induced nmda current after 30 min of recording was significantly lower than the control recording of ifn-induced nmda currents recorded at the start of patch clamping . (b) bar graphs show the average rate of ifn-induced enhancement of nmda current in the same neuron at the beginning of the recording and after 30 min . The value of the right bar is 78.7 5.1% of the left bar . This finding suggested that the ifn-induced enhancement of nmda currents involves the activation of g protein - coupled receptors . * ifn: interferon - gamma; nmda: n - methyl - d - aspartate . The enhancement of nmda - induced inward current by ifn was mediated by ccr2 . (a) bath application of the ccr2 antagonist teijin compound 1 hydrochloride (10 m, 5.5 min) blocked the ifn-induced enhancement of nmda currents . (b) the peak amplitude of the nmda - induced currents after simultaneous application of teijin compound 1 hydrochloride and ifn was 95.0 5.3% that of baseline . Ifn: interferon - gamma; nmda: n - methyl - d - aspartate; n.s . (a) perfusion of spinal cord slices with ccl2 (100 ng / ml, 2.5 min) increased nmda - induced inward currents . (b) the peak amplitude of the nmda - induced currents after simultaneous ccl2 application is 128.3 6.4% compared with baseline . This suggests that ifn increases the nmda - induced inward current in sg neurons via ccl2/ccr2 signaling . * mcp-1: monocyte chemoattractant protein 1; nmda: n - methyl - d - aspartate . In the present study, ifn significantly enhanced nmda - induced inward currents but not ampa - induced inward currents in sg neurons . This ifn-induced facilitation of nmda current was inhibited by an ifn receptor - selective antagonist . The results suggest that ifn enhances nmda receptor signaling by activating ifn receptors in the dorsal horn of the spinal cord . Minocycline, an inhibitor of microglial activation, inhibited the ifn-induced facilitation of nmda current, but inhibition of the neuronal jak - stat signaling did not . Therefore, we conclude that ifn binds its own receptors in microglia but not sg neurons, and the subsequent microglial activation leads to nmda receptor activation . Moreover, ifn-induced enhancement of nmda currents was blocked by gdp--s in the pipette solution or bath application of a ccr2 antagonist . Ccr2 signaling is mediated by a g protein and a typical chemokine involved in central sensitization . These results suggested that activation of ifn receptors in the spinal microglia leads to ccl2 release and subsequently enhances nmda receptors in dorsal horn neurons (figure 8). When nerve injury occurs, ifn levels are increased in the dorsal horn of the spinal cord and binds to ifn receptors on microglia . Ccl2 subsequently enhances the neuronal nmda - induced inward current in lamina ii neurons via ccr2 . Ccl2: chemokine ligand 2; ccr2: c - c chemokine receptor type 2; ifn: interferon - gamma; sg: substantia gelatinosa . When nerve injury occurs, ifn levels are increased in the dorsal horn of the spinal cord and binds to ifn receptors on microglia . Ccl2 subsequently enhances the neuronal nmda - induced inward current in lamina ii neurons via ccr2 . Ccl2: chemokine ligand 2; ccr2: c - c chemokine receptor type 2; ifn: interferon - gamma; sg: substantia gelatinosa . Importantly, this is the first study to investigate the function of ifn using blind whole - cell patch - clamp methods . Interestingly, inward currents induced by exogenously applied nmda, but not ampa, were clearly enhanced by the application of ifn in sg neurons of adult rat spinal cord slices . This postsynaptic effect of ifn on nmda receptor could explain the pain - related behaviors caused by intrathecal ifn. In fact, there is considerable evidence that chronic pain hypersensitivity depends on nmda receptors . It is commonly known that nmda receptor activation is an essential step in initiating and maintaining activity - dependent central sensitization, and it critically contributes to the development of pain hypersensitivity after peripheral tissue damage or nerve injury . On the other hand, no acute changes in ampa - induced current clearly demonstrated that the ifn/ifn receptor system is critical in activating resting spinal microglia, thereby linking them to tactile allodynia . They indicated that spinal microglia are directly stimulated by intrathecal ifn and contribute to ifn receptor - dependent pain hypersensitivity . Our findings also showed that ifn-induced enhancement of nmda currents was microglial ifn receptor - dependent . Notably, ccr2 blockade inhibited the ifn-induced enhancement of nmda - induced inward currents and ccl2 increases nmda current in sg neurons . Demonstrated electrophysiologically that ccl2 can very rapidly (within 2 min) increase nmda currents in dorsal horn neurons . They showed that incubation of the mouse spinal cord slices with 100 ng / ml ccl2 induced substantial activation of extracellular regulated kinase (erk) in lamina i ii, and erk phosphorylation was primarily induced in neun - positive neurons, indicating that ccl2 may directly act on neurons . For instance, exogenous spinal administration of ccl2 induces mechanical allodynia in wildtype but not ccr2 knockout mice, and in a model of neuropathic pain the development of mechanical allodynia is totally abrogated in ccr2 knockout mice . These results are consistent with ours, and in addition, we found that ccl2 lies downstream of ifn in terms of nmda - induced currents . In summary, our results show that ifn enhanced nmda - induced inward currents in sg neurons through the activation of ifn receptors in microglia, and ccl2 was one of the mediators between microglia and neurons . Thus, ifn plays an important role as one of the initiating agents of glia neuron interactions that lead to neuropathic pain due to central sensitization . Ms performed or contributed to all the experiments, analyzed data, and drafted the paper . My, tn, and wt contributed to experiment conception and design and edited the manuscript . The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article . The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was supported in part by mext kakenhi 26462253 to wt, mext kakenhi 25460730 to tn, and mext kakenhi 25462303 to st.
Hemophilia a is a congenital disease transmitted by the x chromosome with a recessive trait, characterized by a deficiency in the production of factor viii . The hemophilic pseudotumor affects 12% of patients with a severe disease, frequently associated with a traumatic injury . The hemophilic pseudotumor develops from repeated episodes of bleeding, either from fracture sites or bleeding or subperiosteal hemorrhage of any soft tissue . The interior of the pseudotumor consists of blood products at different stages of development, surrounded by a fibrous capsule containing hemosiderin - laden macrophages . It appears as a painless tumor of slow growth that can compress key organs causing bone destruction, muscle and skin necrosis . The objective of case presentation is to describe a patient with a hemophilic pseudotumor of the abdomen, and to review the literature on hemophilic pseudotumor . This is a case of a 31 year old male, diagnosed with severe hemophilia a at 2 years of age, treated with factor vii 2000 iu weekly since, whit a family history of 2 cousins with hemophilia (no other specified). In september 2011, he underwent drainage of a hematoma of the left pelvic limb . In october 2011 blood transfusion on multiple occasions . In august 2012 had a hematoma in the left thigh surgically removed . In september 2012 presented upper gastrointestinal bleeding requiring hospitalization with remission of the symptoms . An abdominal tumor diagnosed 10 years ago during routine screening that progressively grew to encompass the entire abdominal area, with no associated symptoms . During his last hospital admission, hematology referred him to general surgery due to bowel obstruction symptoms, and to evaluate surgical treatment of a pelvic tumor referred as a possible teratoma . A ct scan of the abdomen (figs . 1 and 2) was performed, showing a tumor in the pelvis with clear borders, 241 mm 192 mm of diameter, from the pelvis and projected into the bladder, rectum and adjacent structures, including the kidneys . The lesion had irregular wall thickness, with a density of 195 houndsfield units in its interior, with heterogeneous enhancement and vascularity present within; with tomographic diagnosis of abdominal pelvic teratoma . The patient underwent surgery with surgical findings that included a cyst - like tumor of 40 cm 30 cm displacing loops of small bowel, colon and bladder, with adhesions to the great omentum, small intestine, descending colon and bladder . Macroscopically appeared as an old, organized, and calcified clots with a volume of 5000 ml . An abdominal tumor diagnosed 10 years ago during routine screening that progressively grew to encompass the entire abdominal area, with no associated symptoms . During his last hospital admission, hematology referred him to general surgery due to bowel obstruction symptoms, and to evaluate surgical treatment of a pelvic tumor referred as a possible teratoma . A ct scan of the abdomen (figs . 1 and 2) was performed, showing a tumor in the pelvis with clear borders, 241 mm 192 mm of diameter, from the pelvis and projected into the bladder, rectum and adjacent structures, including the kidneys . The lesion had irregular wall thickness, with a density of 195 houndsfield units in its interior, with heterogeneous enhancement and vascularity present within; with tomographic diagnosis of abdominal pelvic teratoma . The patient underwent surgery with surgical findings that included a cyst - like tumor of 40 cm 30 cm displacing loops of small bowel, colon and bladder, with adhesions to the great omentum, small intestine, descending colon and bladder . Macroscopically appeared as an old, organized, and calcified clots with a volume of 5000 ml . The hemophilic pseudotumor of the abdomen is a rare but often disabling condition, potentially fatal in patients with severe hemophilia . Diagnosing a hemophilic pseudotumor with invasive techniques such as, aspiration and biopsy imaging techniques of which mri is preferred, allows recognition of blood products in various stages of evolution . Ultrasonography (usg) shows a central anechoic region with increased echoes behind the lesion due to enclosed fluid in the pseudotumor . Computed tomography (ct) identifies the thick pseudocapsule, but cannot differentiate a hematoma from a chronic abscess . Ct is particularly helpful in the evaluation of bone, whereas mri is superior to ct for delineating soft tissue . At the moment, however, there are instances where surgical extraction of the lesion is not feasible . In such situations, the decision to operate on this patient was made based on the gradual increase in the tumor size, the symptoms that the displayed by the patient . In this patient this type of procedure is associated with complications such as bleeding, bowel perforation, and damage to nearby structures due to firm vascular, and nervous adhesions . Surgical resection after performing arterial embolization to reduce the vascularization of the pseudotumor is a good alternative, thereby reducing the size of the pseudotumor and the risk of bleeding complications during surgery, at best about 2 weeks prior to surgery . It is a rare pathological entity, but it must be considered in the hemophilic patient with a long - standing abdominal tumor and trauma history, to plan appropriate treatment . The abdominal hemophilic pseudotumor is a rare pathological entity, with few reports worldwide, but must be considered in hemophilic patients with a well documented abdominal tumor, as a complication of the hematologic disease . Signs and symptoms of compression should be evaluated by ct or mri because its early diagnosis is crucial for evaluation and proper surgical planning, and treatment in conjunction with other specialties . Written informed consent was obtained from the patient for publication of this case report and case series and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request . The study conception and design, as well as writing the manuscript works were all equally distributed among all the authors.key learning pointshemophilic pseudotumor etiology.hemophilic pseudotumor management.
Approximately, 1.6 million new cases of lung cancer are diagnosed each year throughout the world . Since carcinogenesis is caused by the stepwise accumulation of genetic changes, it is very important to understand the alterations of chromosome and gene in cancer cells . The traditional giemsa banding (g - banding) technique is applied to detect cell sample with a higher mitotic index, but lung cancer cells have a lower mitotic index, which makes karyotypic analysis difficult . In our study, combining with the latest molecular biology techniques, we investigated the chromosomal and genetic alterations in the human lung adenocarcinoma cell line om . Here, we summarized the chromosomal and genetic abnormalities detected in lung cancer cells and discussed the possible implication of those alterations in the processes of tumorigenesis . The lung adenocarcinoma cell line om was established in the second department of biology kochi medical school, kochi university, japan . The cells in cell line om were from human lung adenocarcinoma tissues . After cultured and purified, these cells developed into a special cell line for cancer research . The cultures were harvested and g - banding of the chromosomes was performed by hypo - osmolality, fixation, trypsinization, and giemsa (invitrogen corp ., carlsbad, ca, usa) staining . Samples were evaluated, and images were captured using an olympus bx60 fluorescence microscope (olympus, tokyo, japan) and analyzed with the aid of image analysis software (cytovision, applied imaging corp ., santa clara, ca, usa). After washing with ethanol, dried, concentrated, and purified, the sample and reference genomic dnas were digested and random primed labeled with cyanine-5/cyanine 3-dutp (vysis corp . Cohybridization of these dnas to a comparative genomic hybridization (cgh) arrays was performed for 72 h at 37c ., japan) and data were extracted and analyzed using multiscan medical image analysis system (sony corp ., tokyo, japan). Extracted dna from a normal human was used as the internal control . All the primers were provided by the invitrogen corp ., (invitrogen corp ., carlsbad, ca, usa). The amplified samples were analyzed by electrophoresis at 100 v on 1.5% agarose gels stained with ethidium bromide . The visual of the bands of amplified dna were captured by a camera in the device ., cary, nc, usa) is useful statistical software to analyze this kind of data . An exact 95% confidence interval (ci) using the clopper pearson method was given for the positive rate of abnormal chromosomes and genes . The lung adenocarcinoma cell line om was established in the second department of biology kochi medical school, kochi university, japan . The cells in cell line om were from human lung adenocarcinoma tissues . After cultured and purified, these cells developed into a special cell line for cancer research . The cultures were harvested and g - banding of the chromosomes was performed by hypo - osmolality, fixation, trypsinization, and giemsa (invitrogen corp ., carlsbad, ca, usa) staining . The slides after denaturation were dehydrated in graded series of ethanol and air dried . The 24 xcyte mfish kit (vysis corp ., samples were evaluated, and images were captured using an olympus bx60 fluorescence microscope (olympus, tokyo, japan) and analyzed with the aid of image analysis software (cytovision, applied imaging corp ., santa clara, ca, usa). Dna extraction was performed with 2-propanol (sigma - aldrich, japan). After washing with ethanol, dried, concentrated, and purified, the sample and reference genomic dnas were digested and random primed labeled with cyanine-5/cyanine 3-dutp (vysis corp . Cohybridization of these dnas to a comparative genomic hybridization (cgh) arrays was performed for 72 h at 37c ., japan) and data were extracted and analyzed using multiscan medical image analysis system (sony corp ., all the primers were provided by the invitrogen corp ., (invitrogen corp ., carlsbad, ca, usa). The amplified samples were analyzed by electrophoresis at 100 v on 1.5% agarose gels stained with ethidium bromide . The visual of the bands of amplified dna were captured by a camera in the device ., cary, nc, usa) is useful statistical software to analyze this kind of data . An exact 95% confidence interval (ci) using the clopper pearson method was given for the positive rate of abnormal chromosomes and genes . Characterizations of complex chromosomal abnormalities in the human lung adenocarcinoma cell line om were showed by g - banding analysis, and the wrong number of chromosomes was present in almost all cells . Either aneuploid or polyploid karyotype was observed which displayed chromosome instability in the human lung adenocarcinoma cell line om . G - banding showed chromosomal aberrations on all chromosomes except chromosome x (95% ci: 064%) and 19 (95% ci: 064%) [figure 1]. Giemsa - banding analysis revealed chromosomal aberrations on all chromosomes except, 4, 8, 14, 15, 17, and 19 . The structurally abnormal chromosomes were analyzed by multiplex fluorescence in situ hybridization (m - fish). As was illustrated in figure 2, chromosome 10 is the most frequently (95% ci: 29100%) involved in translocations with six different interchromosomal translocations, and its frequency was 46 times in each sample . The other chromosomes involved in translocation were chromosomes 1, 2, 3, 5, 6, 7, 8, 9, 11, 16, and 18, and the 95% ci minimum value of each statistical datum was more than 9% . Multiple numerical and structural aberrations were observed in the majority of cells from multiplex fluorescence in situ hybridization image . The other chromosomes involved in translocation were chromosomes 2, 5, 9, 11, 18 and 3, 21 . Cgh revealed that the gains in the region of chromosome bands 3q25.3 - 28, 5p13, 12q22 - 23.24, and the losses in that of 3p25 - 26, 6p25, 6q26 - 27, 7q34 - 36, 8p22 - 23, 9p21 - 24, 10q25 - 26.3, 12p13.31 - 13.33, and 17p13.1 - 13.3 [figure 3]. Array comparative genomic hybridization analysis showed partial deletion of in 3p24.1 - 26, 6p25, 6q25.2 - 27, 7q32 - 36, 8p22 - 23, 9p21 - 24, 10q25 - 26.3, 12p13.31 - 13.33, and 17p131 - 13.3 together with duplication at 3q24 - 28, 5p13, and 12q22 - 23.24 . According to the results obtained by cgh the results of the polymerase chain reaction (pcr) experiments showed that the genes membrane metalloendopeptidase (mme) (95% ci: 188%), sucrase - isomaltase (si) (95% ci: 54100%), butyrylcholinesterase (bche) (95% ci: 16100%), and kininogen (kng) (95% ci: 172%) were amplified in this region . However, the amplification of genes histidine - rich glycoprotein (hrg, 95% ci: 060%) and mucin (95% ci: 052%) were not observed [figure 4]. Agarose gel electrophoresis of polymerase chain reaction amplification products from the human lung adenocarcinoma cell line om . N: negative control (normal human lung cell line); om: human lung adenocarcinoma cell line om . Mme: membrane metalloendopeptidase; si: sucrase - isomaltase; bche: butyrylcholinesterase; kng: kininogen; hrg: histidine - rich glycoprotein; muc: mucin . Characterizations of complex chromosomal abnormalities in the human lung adenocarcinoma cell line om were showed by g - banding analysis, and the wrong number of chromosomes was present in almost all cells . Either aneuploid or polyploid karyotype was observed which displayed chromosome instability in the human lung adenocarcinoma cell line om . G - banding showed chromosomal aberrations on all chromosomes except chromosome x (95% ci: 064%) and 19 (95% ci: 064%) [figure 1]. Giemsa - banding analysis revealed chromosomal aberrations on all chromosomes except, 4, 8, 14, 15, 17, and 19 . The structurally abnormal chromosomes were analyzed by multiplex fluorescence in situ hybridization (m - fish). As was illustrated in figure 2, chromosome 10 is the most frequently (95% ci: 29100%) involved in translocations with six different interchromosomal translocations, and its frequency was 46 times in each sample . The other chromosomes involved in translocation were chromosomes 1, 2, 3, 5, 6, 7, 8, 9, 11, 16, and 18, and the 95% ci minimum value of each statistical datum was more than 9% . Multiple numerical and structural aberrations were observed in the majority of cells from multiplex fluorescence in situ hybridization image . The other chromosomes involved in translocation were chromosomes 2, 5, 9, 11, 18 and 3, 21 . Cgh revealed that the gains in the region of chromosome bands 3q25.3 - 28, 5p13, 12q22 - 23.24, and the losses in that of 3p25 - 26, 6p25, 6q26 - 27, 7q34 - 36, 8p22 - 23, 9p21 - 24, 10q25 - 26.3, 12p13.31 - 13.33, and 17p13.1 - 13.3 [figure 3]. Array comparative genomic hybridization analysis showed partial deletion of in 3p24.1 - 26, 6p25, 6q25.2 - 27, 7q32 - 36, 8p22 - 23, 9p21 - 24, 10q25 - 26.3, 12p13.31 - 13.33, and 17p131 - 13.3 together with duplication at 3q24 - 28, 5p13, and 12q22 - 23.24 . According to the results obtained by cgh, gene analysis was performed in the 3q24 - 28 region . The results of the polymerase chain reaction (pcr) experiments showed that the genes membrane metalloendopeptidase (mme) (95% ci: 188%), sucrase - isomaltase (si) (95% ci: 54100%), butyrylcholinesterase (bche) (95% ci: 16100%), and kininogen (kng) (95% ci: 172%) were amplified in this region . However, the amplification of genes histidine - rich glycoprotein (hrg, 95% ci: 060%) and mucin (95% ci: 052%) were not observed [figure 4]. Agarose gel electrophoresis of polymerase chain reaction amplification products from the human lung adenocarcinoma cell line om . N: negative control (normal human lung cell line); om: human lung adenocarcinoma cell line om . Mme: membrane metalloendopeptidase; si: sucrase - isomaltase; bche: butyrylcholinesterase; kng: kininogen; hrg: histidine - rich glycoprotein; muc: mucin . Lung cancer remains the leading cause of cancer - related morbidity and mortality worldwide . Unlike in leukemia research, lung cancer cell has a lower cell mitotic index, which makes karyotypic analysis difficult . Using modern genetic technologies, we investigated the chromosome and gene alterations in human lung adenocarcinoma of cell line om . The goal of our study is to identify the alterations of chromosome and gene in human lung adenocarcinoma cell line om, which may provide new insights into the mechanisms of tumorigenesis . With high - resolution g - banding techniques, both the numerical and structural chromosomal abnormalities were observed . G - banding analysis revealed that the changes in chromosome number were present in most cells, and either aneuploidy or polyploidy was identified within them . In om cell line, certain chromosomes frequently participated the structural rearrangement, prominent among them being chromosome 3, 6, 7, 9, 10, 11, 18, and y. although g - banding is a fundamental technique for cytogenetic study, it has limited utility in instances of cryptic or very complex rearrangements . M - fish is a powerful technique that can be used for identifying uniquely all 24 chromosome types of the human genome . The m - fish image showed that the highest frequency of translocation was in chromosome 10 . In genetics, a chromosome translocation is caused by rearrangement of parts between nonhomologous chromosomes, and the distinct classes of chromosomal rearrangements create proto - oncogenes . Loci on chromosome 10 include microseminoprotein beta, which encodes beta - microseminoprotein, a primary constituent of semen and a proposed prostate cancer biomarker . Moreover, multiple tumor - suppressive genes are located in chromosome 10, and the translocations of chromosome 10 may lead to damage of these tumor - suppressive genes . Therefore, frequent translocations of chromosome 10 in cell line om may be catastrophic genomic events and play key roles in tumorigenesis . In general, regional gains on chromosome indicate that oncogenes may exist in this region, while the chromosome losses have been interpreted as an evidence that the region has affected a certain tumor suppressor genes . Cgh analysis revealed that gain regions were observed on chromosomes 3q25.3 - 28, 5p13, and 12q22 - 23.24, which indicated that there may exist oncogenes that were activated in the gain regions . While loss regions were detected in chromosome 3p25 - 26, 6p25, 6q26 - 27, 7q34 - 36, 8p22 - 23, 9p21 - 24, 10q25 - 26.3, 12p13.31 - 13.33, and 17p13.1 - 13.3, which indicated that some tumor suppressor genes may be located in these regions . It is well - known that tumor suppressor genes p16 and p53 were located in chromosome 9p21 and 17p13, respectively . Based on the above results, we detected amplification of genes at chromosome band 3q24 - 28 by pcr in the cell lines . The genes bche, mme, kng, and si were amplified in the chromosomal region . The bche gene provides instructions for making the pseudocholinesterase enzyme, which has a variety of physiological effects such as the metabolism of the cocaine and heroin and the breakdown of organophosphorus esters . Recent studies indicated that bche gene was amplified in the cells of lung carcinomas, which were consistent with our results . Observed the amplification of genes bche and slc2a2 at 3q26 in squamous cell lung carcinomas, whereas no amplification has been found in genes mme and kng . Mme gene is expressed in both normal and neoplastic cells as a common acute leukemia antigen . In squamous cell lung carcinoma, the high mme gene expression was significantly associated with poor overall survival . There were many studies on the expression of mme gene in lung cancer cells; however, these results were quite conflicting . Many factors may be responsible for the difference among the results, such as experimental conditions, experimental methods, and pathological types of lung cancer . The kng gene, which is one of the members of a cystatin - like superfamily, has potential roles in angiogenesis and (or) tumor development . Kinins that derived from kng involved in cell proliferation, leukocyte activation, cell migration, endothelial cell activation, and nociception . Thought that kng may be a novel therapeutic target in chronic lymphocytic leukemia and the possible association with prognosis . We observed that kng gene was amplified in lung adenocarcinoma cell line om, which suggested that kng gene was likely to be involved in tumorigenesis of lung cancer cells . Although there are numerous studies elucidating the role of si gene in the digestion of dietary carbohydrates, there are limited data indicating a specific involvement of si gene in the genesis of human cancers . Our experiments demonstrated that si gene was amplified on chromosome band 3q24 - 28 . Since cancer is attributed to multi - genetic alterations accumulated in the cells, we speculated si gene may be involved in tumorigenesis.in summary, our findings suggested that most of those examined cells exhibited multiple complex karyotypes in human lung adenocarcinoma cell line om . Chromosome 10 was frequently involved in chromosomal translocation, which may be catastrophic genomic events and play key roles in tumorigenesis . According to the results of cgh, we speculated that the oncogenes may be located at 3q25.3 - 28, 5p13, and 12q22 - 23.24, while tumor suppressor genes may exist in 3p25 - 26, 6p25, 6q26 - 27, 7q34 - 36, 8p22 - 23, 9p21 - 24, 10q25 - 26.3, 12p13.31 - 13.33, and 17p13.1 - 13.3 . In human lung adenocarcinoma cell line om, at least four genes (mme, si, bche, and kng) were involved in carcinogenesis.
This may cause inconveniences with regard to activities of daily living; thus physiotherapy is often used to treat problems related to joint movement limitation . In previous studies, we have reported the histopathological changes that occur in the rat knee joint components during immobilization; however, the changes have not been fully clarified . Joint immobilization induces muscle atrophy, articular cartilage loss, and proliferation of connective tissue within the joint space1, 7 . Immobilization of joints surrounded by edematous soft tissue often produces joint stiffness more quickly and more severely than immobilization of nonedematous extremities8 . After two weeks of joint immobilization, the number of synoviocytes in the posterior synovial intima was increased compared with a control group9 . Watanabe reported atrophy of fat cells, proliferation of fibroblasts, and narrowing of the spaces between collagen fibers in the rat knee joint after immobilization10 . Some studies reported changes in joint components caused by remobilization after immobilization8, 11,12,13,14,15 . The range of motion of rat knees immobilized for 8 weeks remained substantially reduced after a 4-week period of unassisted remobilization16 . But histopathological findings of the joint components were not described in that study . In the precedent studies, histopathological changes in joint components after remobilization and/or reloading during joint contracture were observed . A few studies have reported the effect of exercise during an immobilization period17, 18 . Tatsuta related that exercise during immobilization periods did not improve range of motion but also that granulation tissue did not infiltrate into the articular cavity19 . In this study, we used external fixation in order to immobilize the joint and subjected the rats to exercise during the immobilization period . The purpose of this study was to clarify the effects of the rom exercise on joint components according to histopathological analysis . The protocol for these experiments were approved by the animal care committee and institutional ethics committee of kanazawa university, and all procedures for animal care and treatment were performed in accordance with the guidelines for the care and use of laboratory animals at kanazawa university . In total, twenty - six 9-week - old adult male wistar rats (weighing 250275 g) were used in this study . The rats were randomly divided into three groups, the immobilization group (n=10), exercise group (n=10), and control group (n=6). The animals were kept under normal conditions for one week before the start of experiments in order to acclimatize them to the environment . They were housed, 1 per cage, in a room maintained under a 12-hour light - dark cycle . The immobilization group and exercise group were anaesthetized (pentobarbital sodium, 40 mg / kg bw, ip) and operated on under sterile conditions . The right knee joints of the rats in the immobilization group and exercise group were immobilized with external fixation at 120 degrees of flexion . A 2-mm longitudinal incision was made in the right hind thigh skin; the right femur and tibia were pierced by kirschner wire with a diameter of 0.8 mm, and the wire was then bent (fig . The right femur and tibia were pierced by kirschner wire with a diameter of 0.8 mm, and the wire was then bent (x - ray).). Kirschner wires were fixed outside the wounds in a knee flexion posture with screw (4 mm diameter) and nuts . The instruments for external fixation weighed 6 to 7 g. the rat s knee flexion angle (120 degrees) was determined by the method described in our previous studys13, 21, 22 . This immobilization method is low cost, and immobilization can be reversed to perform exercise and then reestablished easily . Although the right hindlimb knee joint was immobilized, the rats were able to move freely without restriction in their respective cages and had free access to food and water . The right femur and tibia were pierced by kirschner wire with a diameter of 0.8 mm, and the wire was then bent (x - ray). Range of motion exercise (rom ex) was started from the day after immobilization . Rom ex was performed in the exercise group once a day for 3 minutes, 6 days a week, for 2 weeks . The mean torque necessary for the rat to passively extend the knee joint was 1 n, and this value was adopted as the strength during the rom ex . The exercise consisted in 18 cycles of 10 seconds of exercise (a total of 3 minutes). The exercise time was determinate according to the results of a preliminary experiment . In the first 5 seconds of the exercise cycle, the rat s right hind limb was maintained in 120 degrees of hip flexion, and in the next 5 seconds, the limb was pulled in the caudal direction with 1 n force applied by a manual force gauge . The rom exercise intensity was controlled using a force gauge continuously during the 3 minutes . After each exercise, the rat knee was re - immobilized by external fixation at 120 degrees of knee flexion . The control group did not undergo surgery and was not subjected to exercises . After 2 weeks of intervention, the roms of all rat knees (extension limitation) were measured under anaesthesia . In the rats in the immobilization and exercise groups, the external fixation was removed, and shortly after the tight hind limbs of the rats were later pulled with 1 n force in the caudal direction . After angle measurement, the knees of the rats were re - immobilized at 120 degrees of flexion by external fixation . Later, the rats were sacrificed by intraperitoneal injection of an overdose of pentobarbital sodium . Immediately after euthanasia, their right hind limbs the right knees of the rats were fixed in 10% buffered formalin and decalcified . After decalcification, they knees were cut in the sagittal plane at the level of the anterior and posterior cruciate ligament . Following neutralization with 5% sodium sulfate solution, the specimens were fixed, decalcified, and neutralized at 4 c for 72 hours . The specimens were embedded in paraffin, sectioned at 3 m, and mounted on microscope slides . All sections were stained with hematoxylin and eosin and toluidine blue stains and used for observation in a histopathological examination . Histopathological analysis was performed by examining the synovial membrane, the joint capsule, and the joint cartilage . Knee limitation data were statistically analyzed using ibm spss statistics for windows (version 19.0.1, ibm corp . Rom was analyzed using one - factor analysis of variance (anova) with bonferroni post - hoc multiple comparisons . The histopathological analysis showed no swelling or signs of infection in the knees as a result of the intervention . The mean values of knee rom (extension limitation angle) were 19.33.0 degrees, 77.37.4 degrees, and 51.04.9 degrees in the control group, immobilization group, and exercise group, respectively, and each groups showed a statistically significant difference (p<0.05). The mean body weights were 326.08.8 g, 310.616.6 g, and 304.57.7 g in the control group, immobilization group, and exercise group, respectively . There were statistically significant differences (p<0.05) between the control group and other groups . In the control group, the joint capsule was typically composed of coarse and relatively loose fibrous connective tissues . The joint capsule in the immobilization group and exercise group showed narrowing of the collagen bundles in interstitial spaces but was less dense in the control group (fig . The joint capsule in the control group was typically composed of coarse and relatively loose fibrous connective tissues, but those in the immobilization group and exercise group showed narrowing of the collagen bundles of interstitial spaces; the joint capsule was less dense in the control group . A, control group; b, immobilization group; c, exercise group . The surface of the articular cartilage in animals of the control group was smooth, and the hyaline cartilage was directly exposed to the articular cavity (fig . The surface of the articular cartilage was smooth, and the hyaline cartilage was directly exposed to the articular cavity (a); the cartilage matrix of the hyaline cartilage was stained with toluidine blue (b). F, femur; t, tibia; m, meniscus . He stain 200). The cartilage matrix of the hyaline cartilage was stained with toluidine blue . In the immobilized group, the articular cartilages were covered with the proliferating tissue composed of fibroblast - like spindle - shaped cells (fig . 4 . Hyperplastic tissues infiltrated into the articular cavity and adhered to the surface of the articular cartilage (white arrow head), and infiltration of vessels (black arrows) was observed . Conversely, in the exercise group, hyperplasia of membrane - like tissue was localized to the synovial membrane, and the infiltration of fibroblast - like spindle - shaped cells was localized to the articular cavity (fig . 5 . Hyperplasia of tissue was localized to the meniscus (black arrows), and the surface of the articular cartilage was smooth (a). The cartilage matrix of the hyaline cartilage was stained with toluidine blue (b). Notably, the presence of erythrocytes that had leaked into the articular cavity was observed in some exercise group animals (n=6), suggesting the presence of hemorrhage after rom ex . The joint capsule in the control group was typically composed of coarse and relatively loose fibrous connective tissues, but those in the immobilization group and exercise group showed narrowing of the collagen bundles of interstitial spaces; the joint capsule was less dense in the control group . A, control group; b, immobilization group; c, exercise group . The surface of the articular cartilage was smooth, and the hyaline cartilage was directly exposed to the articular cavity (a); the cartilage matrix of the hyaline cartilage was stained with toluidine blue (b). He stain 200 hyperplastic tissues infiltrated into the articular cavity and adhered to the surface of the articular cartilage (white arrow head), and infiltration of vessels (black arrows) was observed . He stain (a), toluidine blue stain (b) 200 hyperplasia of tissue was localized to the meniscus (black arrows), and the surface of the articular cartilage was smooth (a). The cartilage matrix of the hyaline cartilage was stained with toluidine blue (b). Proliferation of intracapsular connective tissue and the formation of adhesions are primary responses to limitation of motion5 . In this study, we observed the presence of granulation tissue - like organization and infiltration in the joint cavity after two weeks of knee immobilization in rats . The cartilage appeared to be more or less confluent with the overlying connective tissue20 . These findings are consistent with those reported in our previous study21, 22 . During the first two weeks of immobilization, rom limitation caused damage in the myogenic component, and after two weeks, the arthrogenic component constituted more than 80% of the total restriction in rom20 . This suggests that in our contracture model, the myogenic factor was stronger than the arthrogenic factor for rom limitation . Rom limitation was significantly decreased by exercise, presumably due to its property that contributes to the maintenance of muscle extensibility . However, changes were found not only in the muscular component but also in the joint component . Proliferation of intracapsular connective tissue and the formation of adhesions are primary responses to limitation of motion20 . In the present study, extensive hyperplasia of fibroblasts was observed in all animals in the immobilization group, but it was less severe and was focally distributed in the animals in the exercise group . This finding may suggest that rom exercise induces some change within the joint components and tissue metabolism . Connective - tissue proliferation was present in all three of the knees immobilized for fifteen days, and it was well established at thirty days20 . The changes in the joint capsule were observed from the early period, and there were no important differences between the immobilized group and exercised group . In our model, we did not observe a significant influence of exercise on the joint capsule; however we cannot rule out that some changes may be observe in response to other exercise methods . We observed slight intra - articular hemorrhage, which indicates that the exercises used in this study might cause laceration of granulation tissue and adhesion . In this study, it was clarified that rom ex maintains range of motion and reduces the changes in the joint components . Therefore, it is necessary to examine several kinds of frequency and the exercise strength in a future study . A number of studies have performed remobilization following a joint immobilization period, and some have subjected animals to exercise during an immobilization period.
Thalassemia is a genetic disease with an autosomal recessive inheritance pattern in which the generation of one or more hemoglobin chains have been dropped or completely inhibited . Patients with thalassemia major need regular monthly blood transfusions starting at an age of approximately 6 months to maintain oxygen supply to tissues due to severe anemia . Followed by ineffective hematopoiesis, patients with thalassemia often have high iron absorption from the gastrointestinal tract, and despite iron overload, continue to have a greater intestinal absorption . On the other hand, regular blood transfusions cause iron overload in patient s organs . Through the formation of free radicals and lipid peroxidation, iron overload causes damage to transferrin receptors on the cell surface and some cellular organelles, which disrupts their tolerance and viability, compared to non - organic iron . When levels of total body iron reach 40 g, the function of body organs are disrupted, and at the level of 60 g or more, irreversible heart failure occurs . The major cause of mortality and morbidity in these patients is iron overload in parenchymal tissues . Most patients with thalassemia major will die between ages 16 and 24 due to iron overload, and almost all deaths occur due to iron overload in the heart muscle . The chronic iron overload also causes disturbances in endocrine glands, including the pancreas, pituitary, and thyroid systems . Thalassemia is the most common genetic disease in the world and has a high prevalence in iran as well . The highest prevalence of the disease has observed along the margin of the caspian sea and persian gulf and the provinces of mazandaran and fars (17). Khuzestan province and the populated city of ahvaz are of particular interest because of a variety of factors, including the arab and bakhtiari tribes and other ethnic groups present, geographic location along the persian gulf, tropical weather close to the equator, communication by land and waterways with different regions and numerous wars in this area . This locality can be considered as a large and diverse pool for observing thalassemia and other hemoglobinopathies (8). The number of patients with major thalassemia in khuzestan province has been estimated to be 2,500 patients (9). Primary hypothyroidism is one of the most common side effects seen in patients with thalassemia . Cases of hypothyroidism in patients with major thalassemia in different studies, regardless of incidence, have been reported between 13% and 60% . The prevalence in shiraz, tabriz, and tehran have been reported, respectively, as 7%, 16%, and 7.7% . However, the prevalence has been reported as 21.6% in italy (1013). Increased tsh hormone (thyroid stimulating hormone) with a corresponding decrease in t4, is considered as severe hypothyroidism (1, 14). Additional deposits of iron overload in patients with thalassemia are the major cause of subclinical hypothyroidism early in the first phase and eventually leads to obvious hypothyroidism, which is followed by symptoms, including poor growth, obesity, constipation, decreased energy, increased sleep time, and delayed sexual maturity, that require immediate treatment with thyroid hormones (4). Multiple and contradictory results have been reported for the effects of iron overload on thyroid gland functioning and results vary from normal to abnormal (1517). Thalassemia has a profound impact on the endocrine system function, especially the thyroid and can be studied through its genetic basis and the influence of genetic polymorphisms . Due to the current research contradictions and the high prevalence of thalassemia, regional study of the mechanism of the disease in patients appears to be essential . As a study on this topic has not been completed in the khuzestan province, the aim of this study is to evaluate the levels of thyroid hormones and tsh in patients with beta - thalassemia in the city of ahwaz . This case - control study was conducted in abuzar hospital in the city of ahwaz during 20152016 . The case group included 105 patients with thalassemia that were referred to the treatment center . The control group also consisted of 105 healthy subjects admitted to the same medical center . Before entering the study, inclusion criteria include the absence of infectious diseases, autoimmune diseases, thyroid and parathyroid glands diseases, acute and chronic gastrointestinal diseases, and that any vaccine or surgery was completed over a month before the test . Obviously, the inclusion criteria originated in the review process . In this study, groups were matched for sex and age and analysis was carried out through the selection of controls appropriate to the case . Blood sampling from the patients was prescribed by a doctor and the required tests were completed after permission from the patients . Recognition and inclusion of patients in the case group was performed based on hemoglobin electrophoresis and all patients were under the medical supervision of a professor of hematology . Exclusion criteria included a diagnosis of thalassemia minor (mild or moderate), splenectomy, family history of hypothyroidism, thyroid - stimulating drug consumption and the risk of acute diseases, or oncology diseases . From all persons in the case and control groups, fasting blood samples were taken at 8 am for measurement of t3, t4, and tsh . Serum levels of hormones were measured by the enzyme - linked immunosorbent assay method and with the help of a stat fax 2100 . In order to measure hormone concentrations of t3, t4, and tsh, a kit of mono bind was used with a sensitivity 0.12 (ng / ml), 0.4 (g / dl), and 0.078 (iu / ml). Chicago, illinois, usa) and was analyzed using independent t - test and logistic regression analysis . In this study, the case and control groups were the same with regards to age and gender . Regarding gender, the subjects distributions were similar in both groups and all studied subjects were in the age range of 1520 years . For comparison, the average of three variable, t3, t4 and tsh from the two groups of case and control and the two independent sample t - test was used according to the normal distribution of data . Serum level of t3 hormone in the case and control groups showed no significant difference (p> 0.05). In this study, serum t4 levels were lower in the case group than the control group and the difference was statistically significant (p <0.05). Serum levels of thyroid stimulating hormone (tsh) were also higher in the treatment group than the control group and the difference was statistically significant (p <0.05) (table 1). Logistic regression analysis shows statistically significant differences in serum levels of t4 (or: 0.58, with a 95% confidence level and 0.46 to 0.72 of confidence interval) and tsh (or: 1.57, with a 95% confidence level and 1.22 to 2.01 confidence interval) between the two case and control groups (p <0.05). Interestingly, serum level of t3 in the case and control groups showed no significant difference (p> 0.05) (table 2). In this study, 105 patients with thalassemia major were compared in terms of the thyroid gland functioning with 105 healthy subjects as controls . The serum t4 levels in patients were lower compared to the control group, while the tsh levels were higher . The logistic regression analysis showed that serum t4 level in the experimental group was 0.58, compared to the control group, and serum tsh level in cases is 1.57 times more than the control group . Despite the high ferritin levels in some studies which showed increased body iron, serum levels of pituitary - thyroid reports, the direct relationship between serum ferritin and a decrease of the performance of the pituitary - thyroid axis has been demonstrated (2224). Evaluation the thyroid function is very important due to its tremendous impact on the body s normal growth in infancy and adolescence, as well as participation in many physiological mechanisms . Consequently, due to this vital function, reduced activity of the gland in patients with thalassemia major can be followed by effects, including reduced growth and mental retardation (16). After gonadal failure, hypothyroidism seems to be the most common endocrine disorder in patients with thalassemia major (25). Studies on the thyroid function of patients with thalassemia major have had different results (16). In the jain study on 25 patients with thalassemia major, the serum levels of t3, t4, and tsh were reported as lower for t3 and t4 and higher for tsh compared to healthy subjects (control group) (17). In two other studies, hypothyroidism was observed in none of the patients with thalassemia major (20, 22). These differences in various reports originate from the fact that the prevalence of hypothyroidism varies widely, depending on age, region and type of treatment, which includes the rate of blood collection in a month, chelation therapy, and follow - up intervals of studied patients . The reason for its prevalence involves lack of treatment and poor follow - ups of the disease early in life, which lead to irreversible tissue damage due to excess iron overload (10, 11). Iron poisoning of the thyroid gland may be one of the main causes of hypothyroidism . Also, the effects of iron poisoning on other endocrine glands, such as the gonads, may alter thyroid gland function (26). However, comparing serum ferritin levels in patients with hypothyroidism with the control group showed no logical connection between the current level of serum ferritin and thyroid dysfunction . This is probably due to the fact the serum ferritin levels show the iron overload for the last 3 months, while developing endocrine disorders needs long - term exposure to extra iron (11, 27). Therefore, one can conclude that observations of hypothyroidism among these patients may occur due to a chronic increase of iron and its effect on the thyroid gland, followed by a decline in t3 and t4 levels . In addition to the increased toxicity of iron overload in the body, the hypoxia - induced disorder should also be considered . Previous studies have indicated that blood level of hemoglobin in patients with thalassemia is approximately 7 to 8 grams per deciliter and a high concentration of 2, 3-bisphosphoglycerate and high levels of hbf may lead to tissue hypoxia, which can have a negative effect on thyroid gland function (24). This finding could also explain the reduced activity of the thyroid gland, or in other words, the hypothyroidism in patients with thalassemia major in whom hypoxia is observed as a result of the disease . . These findings double the importance of the annual assessment of thyroid function and early diagnosis in patients with beta thalassemia . With regard to the likely effect of iron on this disorder, a blood transfusion program should be prescribed in shorter intervals so that the concentration of blood hemoglobin stays at approximately 11 g per deciliter . Also, desferal should be regularly injected to prevent hemochromatosis in patients with thalassemia major . Therefore, it is suggested that the iron intake would not exceed the amount of 200 g per kg of patient body weight per year to maintain ferritin concentrations at normal levels . While this study was completed at the center of the province, the findings shown here are relevant on a larger scale.
Actuators, or artificial muscles, based on conducting polymers (cps), and other electrochemically active organic and carbonbased materials, constitute the base for the development of promising polymeric sensing motors, tools for surgeons, or zoomorphic and anthropomorphic soft robots.1, 2, 3, 4, 5, 6, 7, 8, 9, 10 they are electrochemical devices; the cp oxidation / reduction drives the exchange of ions and solvent with the electrolyte causing reversible volume variations and device actuation . Consequently, they are very reliable faradaic motors; the rate of the movement is a linear function of the reaction driving current and the amplitude of the bending movement is under linear control of the consumed charge.11, 12, 13, 14 in addition, the fabrication methodology is very robust; by using different devices (different shapes, including different masses of the cp and fabricated during several years), the same actuation charge per unit of polymer mass always generates the same displacement.11, 13 the exchange of ions is controlled by the driven electrochemical reactions . The exchange of solvent is driven by physical osmotic or electroosmotic effect following, or driven by, respectively, the ionic exchange.15, 16, 17 as a consequence of the delay between faradaic and osmotic effects, the coulodynamic responses show some hysteresis; for the same charge, the muscle position during the forward (oxidation) and return (reduction) is different (actuation hysteresis cycle or dynamical hysteresis). In addition, some devices present creeping effects; after an oxidation / reduction cycle some devices do not recover the initial position.18, 19, 20, 21, 22, 23, 24, 25, 26, 27 recently, irreversible reactions taking place in some cps were identified as one of the creepingeffect origins (chemical creeping).28 by changing the solvent, some cps move from a reactiondriven exchange of cations to reactiondriven exchange of anions.29, 30, 31 by changing the salt (nabr and nacl) in aqueous solutions, the ppy hpps / tape (ppy hpps: polypyrrole paraphenolsulfonic acid blend), the muscle moves from reactiondriven exchange of br to a consecutive exchange of cations (na) and anions (cl) in nacl.33 those results should indicate that the muscle actuation, and the reactiondriven ionic exchange in cps, is conditioned by the variation of the intra and intermolecular forces inside the polymer under the influence of different physical or chemical variables . By changing the solvent or the salt, the dielectric constant, permittivity, dipolar moments, polymerion interactions, or salt dissociation degrees in both the dense polymer gel and the electrolyte should change, altering the ionic exchanges and the concomitant actuation mechanism . Herein, we present the attained results from a different salt, that is, napf6 aqueous solutions, in which the anion is closer to br from a chemical point of view . A comparative study between an asymmetric bilayer muscle (comprised of two different cps) and the three studied bilayer muscles are represented here as: tape / ppy dbs, ppy hpps / tape, and ppy hpps / ppy dbs, indicating the relative (left / right) position of each layer component during experiments . Thus, clockwise / anticlockwise bending movements will be attained under the following reactiondriven volume changes: from the tape / ppy dbs bilayer by ppy hpps / tape bilayer by ppy hpps shrinking / swelling, respectively.from the ppy hpps shrinking or ppy dbs swelling (clockwise) versus preponderant ppy hpps swelling or ppy dbs shrinking (anticlockwise). From the tape / ppy dbs bilayer by ppy hpps / tape bilayer by ppy hpps shrinking / swelling, respectively . From the ppy hpps shrinking or ppy dbs swelling (clockwise) versus preponderant ppy hpps swelling or ppy dbs shrinking (anticlockwise). Figures 1 a c show stationary coulovoltammetric (q / e) responses (each closed loop minimum was taken as the charge zero origin) from the tape / ppy dbs, ppy hpps / tape, and ppy hpps / ppy dbs bilayer muscles, respectively, in 0.5 m napf6 aqueous solutions . We used the specific charge (charge consumed by reaction of the mass unit of cp working inside the electrolyte) because, for artificial muscles exchanging anions11 or cations,34 both the rate of the movement and the angular position are linear functions of the driving current or specific charge, respectively, and, therefore, independent of the film thickness or muscle dimensions . Each bilayer q / e stationary response was obtained after consecutive cycles up to the same anodic potential limit of 0.7 v from a different cathodic potential limit, ranging between 0.6 and 2.0 v. positive charge increases describe oxidations, whereas negative charge increases are related to reduction reactions.35 each q / e response is comprised of two different parts: a closed coulovoltammetric loop, for which the charge (maximum minus minimum) increase gives the charge consumed by reversible oxidation / reduction of the ppy films, and an open part on the left side, for which a charge increase from the initial potential of the cycle to the final potential is the charge consumed by irreversible and slow hydrogen evolution from the organic acid (hdbs or the hpps) taking part in the cp.36 a) coulovoltammetric responses from ppy dbs / tape bilayer artificial muscle in napf6 aqueous solutions obtained by increasing the cathodic potential limit (from 0.6 to 2.0 v) to the same anodic potential limit of 0.7 v. the same experiments were repeated for b) ppy from the tape / ppy dbs bilayer (figure 1 a), both reversible (closed loop) and irreversible (open part) charges increase for rising cathodic potential limits . The reversible charge increase from 1 to 11 mc when the cathodic potential limit moves from 0.6 to 2.0 v, indicating that the polymer reduction occurs in the full studied potential range, up to high cathodic potential limits . Irrespective of the cathodic potential limit, most of the voltammetric charge is consumed by the reversible film oxidation and reduction reactions.35 in terms of the q / e response from the ppy hpps / tape bilayer muscles (figure 1 b), the charge of the closed loop (reversible charge) increases (from 4 to 6 mc) when the cathodic potential limit shifts from 0.6 to 1.2 v. then, the reversible charge drops to 3 mc when the potential limit rises to 2.0 v. the irreversible charge always increases with the cathodic potential limit . The asymmetric bilayer shows (figure 1 c) an intermediate behavior; the redox activity of the bilayer increases from 10 to 13 mc and then drops to 12 mc when the cathodic potential limit moves from 0.6 to 1.5 and then to 2.0 v, respectively . The irreversible charge increase is very fast when the cathodic potential limit moves from 1.5 to 2 v. from the video frames recorded during the bilayers bending movement in parallel to the applied potential cycle, between 1.0 and 0.7 v at 10 mv s, the dynamovoltammetric (angle potential, /e) plots were attained (figure 2 a from the tape / ppy dbs bilayer, figure 2 b from the ppy hpps / tape bilayer, and figure 2 c from the ppy the images in figure 2 d show the asymmetric bilayer muscle position at each potential point, as indicated on the q / e response in figure 2 c, and the described angular displacements between the consecutive potential (points on the q / e response). Evolution of the described angles from the different bilayer artificial muscles: a) ppy hpps / ppydbs, b) ppy hpps / tape, and c) ppy dbs / tape artificial muscles in 0.5 m napf6 aqueous solution during voltammetric experiments between 1.0 and 0.7 v at 10 mv s at room temperature . D) images of the ppy hpps / ppy dbs artificial muscles in 0.5 m napf6 aqueous solution . The three muscles present a similar /e behavior, that is, an anticlockwise bending movement during oxidation and clockwise bending movement during reduction . This means that the ppy dbs film from the tape / ppy dbs bilayer shrinks during oxidation and swells during reduction; the electrochemical reaction drives the exchange of na [eq . (1)]:(1)ppy0dbs - nc+nsmgelppyn+dbs - n+nc++ms+ne- where ppy represents the neutral ppy chains, c are the cations exchanged to keep the electroneutrality, s are solvent molecules to keep the osmotic pressure balance inside the polymeric gel (indicated by subindex gel), and ppy represents every oxidized ppy chain after removal of n electrons (e).37 the amplitude of the described angle is only 4. in a previous paper, an angle of 50 was attained in nabr aqueous solutions under analogous potential cycling.32 this indicates a strong influence of the anion, although, according with equation (1), this influence could be neglected . For the ppy hpps / tape bilayer, the oxidationdriven clockwise angular displacement means that the ppy hpps film swells during oxidation, shrinking during reduction to give anticlockwise movement . Therefore, the reversible electrochemical reaction drives the exchange of anions, a, (pf6 here) [eq . (2)]:33 (2)ppy0hpps+na-+msppyn+hppsa - nsmgel again, a strong influence of the anions emerges, when compared with the results attained from nacl aqueous solutions, whereby cations were exchanged at the beginning of the anodic potential sweep and anions at the end of the sweep; for the results obtained from nabr solutions, only the anion is exchanged in the full potential range . As a consequence, the amplitude of the angle described here per cycle was 24 and only 14 in the previous work (cation exchange followed by anion exchanges gives opposed bending movements during the anodic sweep). As a partial conclusion, in napf6 aqueous solution during oxidation, the ppy dbs film shrinks and the ppy hpps film swells . Thus, when the asymmetric ppy hpps / ppy dbs bilayer was checked in napf6 aqueous solution, both films developed a cooperative dynamic effect; when one of the layers swells and pushes the bending bilayer under oxidation, the second layer shrinks through oxidation and trails the muscle giving larger angular displacements . Structural changes and resulting forces reverse during the bilayer reduction (figure 3). As a consequence of this cooperative actuation of the two constitutive layers, the angle described by the asymmetric bilayer per voltammetric cycle is 133, which is more than six times the amplitude described per cycle for the ppy hpps / tape muscle (22) and over 33 times that described by the tape / ppy dbs muscle (4) when cycling in the same potential range . A second consequence of the cooperative actuation of the two layers in napf6 is a very low creeping effect of 0.3 per cycle . Points (2) and (3) correlate with both the dynamovoltammograms shown in figure 2 and the coulodynamic evolution of the described angles shown in figure 4 . In conclusion, owing to the cooperative actuation, when both layers follow complementary volume changes (swelling / shrinking or shrinking / swelling) driven by the same electrochemical reaction, the angular displacement per potential cycle is greater than those attained from each of the ppy hpps / tape or tape / ppy dbs bilayer muscles . By combining figures 1 and 2, the evolution of each muscle position (angle) as a function of the specific consumed charge (coulodynamic evolution) in napf6 aqueous solutions is attained, as depicted in figures 4 a hpps / ppy dbs muscle angular displacement per cycle is 133 (figure 4 c), which is several times higher than those described by the ppy b) or by the ppy dbs / tape bilayer (4 per cycle; figure 4 a) in the same electrolyte . The coulodynamic efficiencies (slopes) result in 8.5, 3.0, and 1.0 mc for the asymmetric bilayer, the ppy hpps / tape bilayer, and the ppy dbs / tape bilayer, respectively . The magnitudes, described angle per cycle, and coulodynamic efficiency quantify the cooperative dynamical effect of the two layers in the asymmetric bilayer muscle . Relationship between the bending angle described for the muscles and the electrical charges consumed during voltammetric experiments studying reactions of different artificial muscles between 1.0 and 0.7 v at 10 mv s at room temperature: a) ppy dbs / tape, b) ppy hpps / tape, and c) ppyhpps / ppy dbs artificial muscles in napf6 aqueous solution . Both anodic and cathodic displacements show a linear dependence on the charge, with a similar slope, corresponding to any faradaic process driven by the reactions given in equations (1) and (2). The charge spent at the beginning and at the end of the oxidation shows a very low efficiency toward bending the muscle . These charges can be related to hydrogen evolution from the film, producing acids, at the most cathodic potentials and to the elimination of protons at the most anodic potentials.36, 38 both reactions result in very low volume variations . In napf6 aqueous solution, all of the studied cp / tape bilayers behave as faradaic motors; the amplitude of the described angle,, is a linear function of the consumed charge, q [eq . (3)]:(3)=0+kq where 0 is the initial angle and k (mc) is a constant dependent on the system (cp, electrolyte, tape, or second cp film). Nevertheless, the actuation of the asymmetric bilayer presents an important hysteresis effect; for the same charge, the muscle positions during the anodic and the cathodic sweeps are quite different . Smaller hysteresis effects were attributed in previous systems to osmotic and electroosmotic physical effects.33, 39 here, important fractions of the involved charge are consumed at the end of the anodic and cathodic sweeps, giving very low angular displacements (figure 4 c), pointing to an important contribution of the irreversible charge fractions detected in figure (1) to the observed hysteresis . The cooperative actuation in the asymmetric bilayer leads to a higher efficiency of the consumed charge describing larger angular displacements per unit of consumed charge, which is almost one order of magnitude higher than those described by each of the cp / tape bilayers . Nevertheless, the use of cp blends including anions from large organic acids results in important hysteresis effects, which means complex control of the motor movement . In the two studied aqueous electrolytes (nacl and napf6), the expansion and contraction of the ppy hpps film dominates the reactiondriven bending movement of the asymmetric bilayer . The ppy dbs oxidation / reduction has a minor influence on the described amplitude of the angular displacement . The resulting cooperative actuation, when both layers follow complementary volume changes (swelling / shrinking or shrinking / swelling) driven by the same reaction, leads to larger described angles per potential cycle than those attained from each of the ppy in addition to the electrochemical chemodynamic results, it is remarkable that, in napf6 aqueous solutions, the ppy hpps film exchanges, under oxidation / reduction, pf6 with the solution for charge balance . In a previous paper, the same bilayer muscle in nacl aqueous solutions exchanged cations at the beginning of the oxidation sweep and anions at the end of the oxidation sweep . We can conclude that only by changing the anion of the dissolved salt (nacl vs. napf6) in ppy this is equivalent to saying that the presence of pf6 in solution modifies, at least, the ppy pps, and pf6 na interaction forces inside the reacting film, moving from weak na pps interactions to strong ones; na balancing the pps anion is not expelled during oxidation, forcing the entrance of pf6, which is required to balance the positive charges generated on the ppy chains [eq (2)]. Cps have been considered, during their electrochemical reactions in liquid electrolytes, as the simplest material models of the intracellular matrix (icm) dense gel of living cells.40 recently, it has also been discovered that by changing the solvent, but keeping the same dissolved salt, also changes the reactiondriven exchange of cations by exchange of anions.29 these results show that cps and bending artificial muscles constitute a good model for the study and quantification of intermolecular forces evolved during reactions in biomimetic reacting dense gels . The three studied bilayer muscles are represented here as: tape / ppy dbs, ppy hpps / tape, and ppy hpps / ppy dbs, indicating the relative (left / right) position of each layer component during experiments . Thus, clockwise / anticlockwise bending movements will be attained under the following reactiondriven volume changes: from the tape / ppy dbs bilayer by ppy hpps / tape bilayer by ppy hpps shrinking / swelling, respectively.from the ppy hpps shrinking or ppy dbs swelling (clockwise) versus preponderant ppy hpps swelling or ppy dbs shrinking (anticlockwise). From the tape / ppy dbs bilayer by ppy hpps / tape bilayer by ppy hpps shrinking / swelling, respectively . From the ppy hpps shrinking or ppy dbs swelling (clockwise) versus preponderant ppy hpps swelling or ppy dbs shrinking (anticlockwise). Figures 1 a c show stationary coulovoltammetric (q / e) responses (each closed loop minimum was taken as the charge zero origin) from the tape / ppy dbs, ppy hpps / tape, and ppy hpps / ppy dbs bilayer muscles, respectively, in 0.5 m napf6 aqueous solutions . We used the specific charge (charge consumed by reaction of the mass unit of cp working inside the electrolyte) because, for artificial muscles exchanging anions11 or cations,34 both the rate of the movement and the angular position are linear functions of the driving current or specific charge, respectively, and, therefore, independent of the film thickness or muscle dimensions . Each bilayer q / e stationary response was obtained after consecutive cycles up to the same anodic potential limit of 0.7 v from a different cathodic potential limit, ranging between 0.6 and 2.0 v. positive charge increases describe oxidations, whereas negative charge increases are related to reduction reactions.35 each q / e response is comprised of two different parts: a closed coulovoltammetric loop, for which the charge (maximum minus minimum) increase gives the charge consumed by reversible oxidation / reduction of the ppy films, and an open part on the left side, for which a charge increase from the initial potential of the cycle to the final potential is the charge consumed by irreversible and slow hydrogen evolution from the organic acid (hdbs or the hpps) taking part in the cp.36 a) coulovoltammetric responses from ppy dbs / tape bilayer artificial muscle in napf6 aqueous solutions obtained by increasing the cathodic potential limit (from 0.6 to 2.0 v) to the same anodic potential limit of 0.7 v. the same experiments were repeated for b) ppy hpps / tape muscle and c) ppyhpps / ppy dbs muscle . From the tape / ppy dbs bilayer (figure 1 a), both reversible (closed loop) and irreversible (open part) charges increase for rising cathodic potential limits . The reversible charge increase from 1 to 11 mc when the cathodic potential limit moves from 0.6 to 2.0 v, indicating that the polymer reduction occurs in the full studied potential range, up to high cathodic potential limits . Irrespective of the cathodic potential limit, most of the voltammetric charge is consumed by the reversible film oxidation and reduction reactions.35 in terms of the q / e response from the ppy hpps / tape bilayer muscles (figure 1 b), the charge of the closed loop (reversible charge) increases (from 4 to 6 mc) when the cathodic potential limit shifts from 0.6 to 1.2 v. then, the reversible charge drops to 3 mc when the potential limit rises to 2.0 v. the irreversible charge always increases with the cathodic potential limit . The asymmetric bilayer shows (figure 1 c) an intermediate behavior; the redox activity of the bilayer increases from 10 to 13 mc and then drops to 12 mc when the cathodic potential limit moves from 0.6 to 1.5 and then to 2.0 v, respectively . The irreversible charge increase is very fast when the cathodic potential limit moves from 1.5 to 2 v. from the video frames recorded during the bilayers bending movement in parallel to the applied potential cycle, between 1.0 and 0.7 v at 10 mv s, the dynamovoltammetric (angle potential, /e) plots were attained (figure 2 a from the tape / ppy dbs bilayer, figure 2 b from the ppy hpps / tape bilayer, and figure 2 c from the ppy hpps / ppy dbs asymmetric bilayer). The images in figure 2 d show the asymmetric bilayer muscle position at each potential point, as indicated on the q / e response in figure 2 c, and the described angular displacements between the consecutive potential (points on the q / e response). Evolution of the described angles from the different bilayer artificial muscles: a) ppy dbs / tape artificial muscles in 0.5 m napf6 aqueous solution during voltammetric experiments between 1.0 and 0.7 v at 10 mv s at room temperature . D) images of the ppy hpps / ppy dbs artificial muscles in 0.5 m napf6 aqueous solution . The three muscles present a similar /e behavior, that is, an anticlockwise bending movement during oxidation and clockwise bending movement during reduction . This means that the ppy dbs film from the tape / ppy dbs bilayer shrinks during oxidation and swells during reduction; the electrochemical reaction drives the exchange of na [eq . (1)]:(1)ppy0dbs - nc+nsmgelppyn+dbs - n+nc++ms+ne- where ppy represents the neutral ppy chains, c are the cations exchanged to keep the electroneutrality, s are solvent molecules to keep the osmotic pressure balance inside the polymeric gel (indicated by subindex gel), and ppy represents every oxidized ppy chain after removal of n electrons (e).37 the amplitude of the described angle is only 4. in a previous paper, an angle of 50 was attained in nabr aqueous solutions under analogous potential cycling.32 this indicates a strong influence of the anion, although, according with equation (1), this influence could be neglected . Hpps / tape bilayer, the oxidationdriven clockwise angular displacement means that the ppy hpps film swells during oxidation, shrinking during reduction to give anticlockwise movement . Therefore, the reversible electrochemical reaction drives the exchange of anions, a, (pf6 here) [eq . (2)]:33 (2)ppy0hpps+na-+msppyn+hppsa - nsmgel again, a strong influence of the anions emerges, when compared with the results attained from nacl aqueous solutions, whereby cations were exchanged at the beginning of the anodic potential sweep and anions at the end of the sweep; for the results obtained from nabr solutions, only the anion is exchanged in the full potential range . As a consequence, the amplitude of the angle described here per cycle was 24 and only 14 in the previous work (cation exchange followed by anion exchanges gives opposed bending movements during the anodic sweep). As a partial conclusion, in napf6 aqueous solution during oxidation, the ppy dbs film shrinks and the ppy hpps film swells . Thus, when the asymmetric ppy hpps / ppy dbs bilayer was checked in napf6 aqueous solution, both films developed a cooperative dynamic effect; when one of the layers swells and pushes the bending bilayer under oxidation, the second layer shrinks through oxidation and trails the muscle giving larger angular displacements . Structural changes and resulting forces reverse during the bilayer reduction (figure 3). As a consequence of this cooperative actuation of the two constitutive layers, the angle described by the asymmetric bilayer per voltammetric cycle is 133, which is more than six times the amplitude described per cycle for the ppy hpps / tape muscle (22) and over 33 times that described by the tape / ppy dbs muscle (4) when cycling in the same potential range . A second consequence of the cooperative actuation of the two layers in napf6 is a very low creeping effect of 0.3 per cycle . Scheme showing the cooperative actuation of the bending ppy hpps / ppy dbs asymmetric bilayer muscle . Points (2) and (3) correlate with both the dynamovoltammograms shown in figure 2 and the coulodynamic evolution of the described angles shown in figure 4 . In conclusion, owing to the cooperative actuation, when both layers follow complementary volume changes (swelling / shrinking or shrinking / swelling) driven by the same electrochemical reaction, the angular displacement per potential cycle is greater than those attained from each of the ppy by combining figures 1 and 2, the evolution of each muscle position (angle) as a function of the specific consumed charge (coulodynamic evolution) in napf6 aqueous solutions is attained, as depicted in figures 4 a c . The ppy hpps / ppy dbs muscle angular displacement per cycle is 133 (figure 4 c), which is several times higher than those described by the ppy hpps / tape bilayer (22 per cycle; figure 4 b) or by the ppy dbs / tape bilayer (4 per cycle; figure 4 a) in the same electrolyte . The coulodynamic efficiencies (slopes) result in 8.5, 3.0, and 1.0 mc for the asymmetric bilayer, the ppy hpps / tape bilayer, and the ppy the magnitudes, described angle per cycle, and coulodynamic efficiency quantify the cooperative dynamical effect of the two layers in the asymmetric bilayer muscle . Relationship between the bending angle described for the muscles and the electrical charges consumed during voltammetric experiments studying reactions of different artificial muscles between 1.0 and 0.7 v at 10 mv s at room temperature: a) ppy hpps / tape, and c) ppyhpps / ppy dbs artificial muscles in napf6 aqueous solution . Both anodic and cathodic displacements show a linear dependence on the charge, with a similar slope, corresponding to any faradaic process driven by the reactions given in equations (1) and (2). The charge spent at the beginning and at the end of the oxidation shows a very low efficiency toward bending the muscle . These charges can be related to hydrogen evolution from the film, producing acids, at the most cathodic potentials and to the elimination of protons at the most anodic potentials.36, 38 both reactions result in very low volume variations . In napf6 aqueous solution, all of the studied cp / tape bilayers behave as faradaic motors; the amplitude of the described angle,, is a linear function of the consumed charge, q [eq . (3)]:(3)=0+kq where 0 is the initial angle and k (mc) is a constant dependent on the system (cp, electrolyte, tape, or second cp film). Nevertheless, the actuation of the asymmetric bilayer presents an important hysteresis effect; for the same charge, the muscle positions during the anodic and the cathodic sweeps are quite different . Smaller hysteresis effects were attributed in previous systems to osmotic and electroosmotic physical effects.33, 39 here, important fractions of the involved charge are consumed at the end of the anodic and cathodic sweeps, giving very low angular displacements (figure 4 c), pointing to an important contribution of the irreversible charge fractions detected in figure (1) to the observed hysteresis . The cooperative actuation in the asymmetric bilayer leads to a higher efficiency of the consumed charge describing larger angular displacements per unit of consumed charge, which is almost one order of magnitude higher than those described by each of the cp / tape bilayers . Nevertheless, the use of cp blends including anions from large organic acids results in important hysteresis effects, which means complex control of the motor movement . In the two studied aqueous electrolytes (nacl and napf6), the expansion and contraction of the ppy hpps film dominates the reactiondriven bending movement of the asymmetric bilayer . The ppy dbs oxidation / reduction has a minor influence on the described amplitude of the angular displacement . The resulting cooperative actuation, when both layers follow complementary volume changes (swelling / shrinking or shrinking / swelling) driven by the same reaction, leads to larger described angles per potential cycle than those attained from each of the ppy in addition to the electrochemical chemodynamic results, it is remarkable that, in napf6 aqueous solutions, the ppy hpps film exchanges, under oxidation / reduction, pf6 with the solution for charge balance . In a previous paper, the same bilayer muscle in nacl aqueous solutions exchanged cations at the beginning of the oxidation sweep and anions at the end of the oxidation sweep . We can conclude that only by changing the anion of the dissolved salt (nacl vs. napf6) in ppy this is equivalent to saying that the presence of pf6 in solution modifies, at least, the ppy pps, and pf6 na interaction forces inside the reacting film, moving from weak na pps interactions to strong ones; na balancing the pps anion is not expelled during oxidation, forcing the entrance of pf6, which is required to balance the positive charges generated on the ppy chains [eq (2)]. Cps have been considered, during their electrochemical reactions in liquid electrolytes, as the simplest material models of the intracellular matrix (icm) dense gel of living cells.40 recently, it has also been discovered that by changing the solvent, but keeping the same dissolved salt, also changes the reactiondriven exchange of cations by exchange of anions.29 these results show that cps and bending artificial muscles constitute a good model for the study and quantification of intermolecular forces evolved during reactions in biomimetic reacting dense gels . We have presented a comparative coulovoltammetric (q / e), dynamovoltammetric (/e), and coulodynamic (q/) study in napf6 aqueous solution of the asymmetric polypyrrole bilayer muscle (ppy hpps / ppy dbs) and the two bilayer muscles tape / ppy hpps and ppy dbs / tape . The q/ responses indicate that, in the tape / ppy hpps bilayer muscle, the actuation originates from reactiondriven exchange anions (pf6), swelling upon oxidation and shrinking upon reduction . The ppy dbs / tape bilayer actuation in napf6 aqueous solution drives the exchange of the cation (na), shrinking upon oxidation and swelling upon reduction . Hpps / ppy dbs asymmetric bilayer muscle, with a cooperative actuation (dynamic synergy) increasing the angle described per unit of charge . High cooperative actuation effects allow the development of more efficient polymeric motors for different medical tools or for the development of zoomorphic and anthropomorphic soft robots . The use of polymeric blends with organic anions or organic salts leads to large hysteresis actuating effects in aqueous solutions; for the same charge, the muscles position is different during the anodic potential sweep compared to that during the cathodic sweep . The presence of hysteresis effects during actuation results in complex control of the motor movement . New families of cps13, 37, 41, 42 must be explored to find the most important cooperative actuations in the absence of these important hysteresis effects, with minimum creeping effects . Reactants, equipment, used electrochemical methodologies, electrogeneration of the cp monolayer and bilayer films, as well as the construction of the cp / tape bilayers were described in previous papers.32, 33 ppy dbs films were cut into 20 mm1 mm strips, each one was 20 m thick and had a mass of 0.532010 mg . A paint (max effect, maxfactor) strip, 6.012.0 mm from the upper border, prevents direct contact between the electrolyte (by capillarity) and the metallic clamp that allows the electrical contact.32, 33 the ppy dbs / tape bilayer muscle inside the solutions was 8 mm long . Hpps films were also cut into 20 mm1 mm strips, each one was 20 m thick and had a mass of 0.137015 mg . Hpps / ppy dbs bilayer films were cut into 15 mm1 mm strips and were 20 m thick and had a mass of 0.3493 mg . Each film was painted with a transversal paint strip on both film sides, 2.07.0 mm of the upper border.
Chronic obstructive pulmonary disease (copd) is a frequent cause of hospital admission and a significant burden to health services worldwide . It is predicted that it will be the third likely leading cause of death globally by 2020 . In england, it is estimated that copd affects 3 million people and accounts for 1.4 million general practice consultations, one million inpatient bed days and over 23,000 deaths each year . Copd is the second most common cause of emergency admission to hospital in england and one of the most costly inpatient conditions treated by the nhs . Copd exacerbations, in particular those requiring hospitalisation, are associated with significant mortality and morbidity . Coughing has been associated with minimum temperature, humidity and wind speed, as well as with shortness of breath with minimum temperature . Exhaled nitric oxide levels, an indicator of airway inflammation, have been associated with lower temperatures . In bavaria, the daily number of ambulatory care visits was associated with temperature, atmospheric pressure and solar radiation, with further associations with humidity and wind speed in north bavaria . Have measured in the laboratory the effect of low temperatures on respiratory health, including bronchoconstriction present in copd patients undergoing hyperventilation with cold air . A recent analysis by hondula et al . Found that, although the association between respiratory health and meteorological variables are complex, models using meteorological parameters may be sufficiently predictive to be used as an early warning system . Indeed, using the uk met office s healthy outlook service, the predictive ability of weather parameters for copd exacerbations has been demonstrated . Some telemedicine interventions and disease management models have reported reductions in hospital admission rates and significant improvements in outcomes of care, whereas others have shown no benefit . The physical exercise component of pulmonary rehabilitation also improves outcomes, although the weather has been reported as a major environmental barrier to improvement . Developed in conjunction with clinicians, healthy outlook is a service delivered by the met office directly to copd patients utilising automated telephone calls; this has been in place since 2006 and ended in 2013 when the service was closed . Periods of higher risk of copd exacerbations are forecast using a rule - based model, combining observed and forecast parameters including season, humidity, temperature, air quality and rates of influenza - like illness . The service provides copd patients with a 10-day advance warning of forthcoming periods of higher risk and encourages them to act proactively to reduce their exposure to risk; in particular, the telephone call asks them to check for early symptoms of an exacerbation and ensure they have sufficient medication . Telephone calls were no more than fortnightly with typically four telephone calls in an average winter . The primary goal of the healthy outlook service is to reduce the severity and length of copd exacerbations, hence improving the quality of life and life expectancy . A number of small studies have been carried out to assess the effectiveness of the healthy outlook service . A multicentre randomised controlled trial concluded that it may help reduce exacerbation rates, but the results were not statistically significant, possibly because of low (78) patient numbers ., salford primary care trust, noted a reduction in visits to general practitioners and out - of - hours services; however, the number of home visits and the overall cost per patient increased . In bradford and airedale, maheswaran et al . Did not find any significant change in hospital admissions associated with the service . Although the primary goal of the healthy outlook service is not necessarily to reduce hospital admission rates, the hospital episodes statistics database has been used to assess the effect of the healthy outlook service on hospital admission rates of all general practitioners that have used the automated service; this is the largest analysis of the effect of the healthy outlook service on hospital admissions and the results are presented here . Between 2007 and 2011, the healthy outlook service was provided to 31,941 copd patients in 661 participating practices in england . A control practice with similar characteristics of deprivation, age profile and rurality the emergency hospital admission rates of copd patients registered in participating practices were compared with those for control practices . The difference, yho, in admission rates per practice between the first year of use of the healthy outlook service and the previous year was calculated . This ensures that any change in hospital admission rate is based on rates that are normalised for the practices using the rate from the previous year as a baseline . This is compared with the difference, yc, in admission rates for the same years for the matched control practice . The difference in differences, yhoyc, was calculated and a paired t - test was used to compare the admission rates between the participating and control practices . This analysis was extended to estimate the effect of the service on the admission rate for the individual participating patient . An enrolled patient is assumed to be fully participating such that their individual difference in admission rate may be regarded as the difference in admission rate of the practice adjusted for the participation rate within the practice . The yhoyc difference was adjusted for patient participation rate by means of the linear regression of yhoyc against the participation rate . The coefficient of this regression provides an estimate of the effect of the healthy outlook service on the individual participating copd patient . The numbers of hospital admissions for each practice and year were obtained from the hospital episode statistics database: these consisted of (1) emergency admissions with a primary diagnosis of copd (icd-10 codes j40 to j44 (ref . 29)) and (2) emergency admissions wherein copd was any one of the diagnosis codes, as the primary diagnosis or as co - morbid copd (termed here as any diagnosis of copd). The years were determined from the start date of the healthy outlook service for each practice, defined as the date the first patient was enrolled: the year before the service is the 1-year period up to the start date, and the first year of the service is the 1-year period from the start date . A hospital admission rate was obtained by dividing the number of admissions by the number of copd patients registered at the practice . The copd 1 indicator reported by practices and available on the quality and outcomes framework database is the ratio of the number (numerator) of patients on the practice s copd register against the total number (denominator) of patients registered at the practice . The copd 1 numerator was extracted from the quality and outcomes framework database and provides the number of copd patients for each practice . The copd 1 indicator is reported at the end of each financial year such that the number of copd patients for the previous year is taken as that last reported before the start date, and the number of patients for the first year is that first reported after the start date (e.g., a start date of 1 december 2008 means that the number of copd patients registered at the end of march 2008 is used for the previous year and that registered at the end of march 2009 is used for the first year). Other factors (e.g., deprivation) may influence the change in admission rates from year to year for any given practice . Therefore, we matched a control practice with similar characteristics to each of the 661 participating practices, by adapting the method developed by the eastern region public health observatory . Each practice was characterised according to their index of multiple deprivation (imd), the age profile of their patients (i.e., the percentage of registered patients aged 65 years or over) and the rurality classification of their middle super - output area given by the office for national statistics . These three parameters were used as percentiles such that the control practices were chosen by finding the lowest total percentile difference between the two groups, such that the nearest available neighbour in terms of these parameters was chosen with replacement for each participating practice . If cimd, cage and cr were the percentiles for imd, age and rurality, respectively, for participating practices, and similarly cimd, cage and cr for the control practices, matched control practices were found by minimising the total percentile difference (\|cimdcimd\|+\|cagecage\|+\|crcr\|). Emergency hospital admission rates for heart failure (icd-10 code i50) and myocardial infarction (icd-10 codes i21 and i22) in the year before the start of the service were compared using paired t - tests, for participating, and control surgeries to ensure that the two groups were well matched . The difference, yho, in admission rates per practice between the first year of use of the healthy outlook service and the previous year was calculated . This ensures that any change in hospital admission rate is based on rates that are normalised for the practices using the rate from the previous year as a baseline . This is compared with the difference, yc, in admission rates for the same years for the matched control practice . The difference in differences, yhoyc, was calculated and a paired t - test was used to compare the admission rates between the participating and control practices . This analysis was extended to estimate the effect of the service on the admission rate for the individual participating patient . An enrolled patient is assumed to be fully participating such that their individual difference in admission rate may be regarded as the difference in admission rate of the practice adjusted for the participation rate within the practice . The yhoyc difference was adjusted for patient participation rate by means of the linear regression of yhoyc against the participation rate . The coefficient of this regression provides an estimate of the effect of the healthy outlook service on the individual participating copd patient . The numbers of hospital admissions for each practice and year were obtained from the hospital episode statistics database: these consisted of (1) emergency admissions with a primary diagnosis of copd (icd-10 codes j40 to j44 (ref . 29)) and (2) emergency admissions wherein copd was any one of the diagnosis codes, as the primary diagnosis or as co - morbid copd (termed here as any diagnosis of copd). The years were determined from the start date of the healthy outlook service for each practice, defined as the date the first patient was enrolled: the year before the service is the 1-year period up to the start date, and the first year of the service is the 1-year period from the start date . A hospital admission rate was obtained by dividing the number of admissions by the number of copd patients registered at the practice . The copd 1 indicator reported by practices and available on the quality and outcomes framework database is the ratio of the number (numerator) of patients on the practice s copd register against the total number (denominator) of patients registered at the practice . The copd 1 numerator was extracted from the quality and outcomes framework database and provides the number of copd patients for each practice . The copd 1 indicator is reported at the end of each financial year such that the number of copd patients for the previous year is taken as that last reported before the start date, and the number of patients for the first year is that first reported after the start date (e.g., a start date of 1 december 2008 means that the number of copd patients registered at the end of march 2008 is used for the previous year and that registered at the end of march 2009 is used for the first year). Other factors (e.g., deprivation) may influence the change in admission rates from year to year for any given practice . Therefore, we matched a control practice with similar characteristics to each of the 661 participating practices, by adapting the method developed by the eastern region public health observatory . Each practice was characterised according to their index of multiple deprivation (imd), the age profile of their patients (i.e., the percentage of registered patients aged 65 years or over) and the rurality classification of their middle super - output area given by the office for national statistics . These three parameters were used as percentiles such that the control practices were chosen by finding the lowest total percentile difference between the two groups, such that the nearest available neighbour in terms of these parameters was chosen with replacement for each participating practice . If cimd, cage and cr were the percentiles for imd, age and rurality, respectively, for participating practices, and similarly cimd, cage and cr for the control practices, matched control practices emergency hospital admission rates for heart failure (icd-10 code i50) and myocardial infarction (icd-10 codes i21 and i22) in the year before the start of the service were compared using paired t - tests, for participating, and control surgeries to ensure that the two groups were well matched . The number of emergency hospital admissions from the hospital episode statistics database and the number of copd patients registered from the quality and outcomes framework database (table 1) provide the admission rates for each participating and control practice before and after the start of the healthy outlook service . The mean patient participation rate in relation to the copd patient population was 40.7% with 95% of practices, with a participation rate between 11 and 81% . Prediction intervals are provided because they are a measure of the scatter and distribution of the data . Histograms of the characteristics for practices participating in healthy outlook and for all general practices in england are compared in figure 1: the plots suggest that the distribution of participating practices does not differ much from that of an average english practice . From matching a control practice to each participating practice, the mean percentile difference between matched practices was 2.3% with 95% of matches with a percentile difference within 0.84.3% . Figure 2 shows a comparison of the imd and age between participating and control practices, the tight clustering of the points along the diagonal suggesting a good match . Paired t - tests of the admission rates for heart failure and myocardial infarction in the year before the start of the service resulted in a mean difference between the two groups of 0.01 (95% confidence interval (ci)=0.08 to 0.07; p=0.9) for heart failure and 0.02 (95% ci=0.09 to 0.05; p=0.6) for myocardial infarction . This suggests that the participating practices are well matched to their controls as there were no differences in emergency hospital admission rates for these two outcomes . For admissions with a primary diagnosis of copd, the mean absolute change in admission rate for participating practices between the start year and the previous year was 1.5% (95% ci=2.2 to 0.8%). For the control practices this figure was 0.8% (95% ci=1.5 to 0.0%). The difference between participating and for admissions with any diagnosis of copd, there was a mean absolute reduction in admission rates for participating practices of 2.0% (95% ci=3.5 to 0.4%) and, a corresponding increase of 0.3% (95% ci=1.3 to 1.9%) in the control practices . The paired t - test yielded a difference, yhoyc, of 2.3% (95% ci=4.2 to 0.4%; p=0.02). For the regression of the yhoyc difference against participation rate, the regression coefficient is 0.023 (95% ci=0.045 to 0.002; p=0.04) for admissions with a primary diagnosis of copd . This equates to an estimated difference in admission rate of 2.3% for the participating copd patient . For admissions with any diagnosis of copd (i.e., when copd was the primary diagnosis or a co - morbid condition), the regression coefficient was 0.07 (95% ci=0.11 to 0.03; p=0.001). This equates to an estimated difference in admission rate of 7% for the participating patient . In the year after recruitment, the difference between participating and control practices was 0.8% for admissions with a primary diagnosis of copd . Therefore, for an average practice with a primary diagnosis admission rate of 15% (see table 2), the effect of the healthy outlook service was a 5% drop in admissions with a primary diagnosis of copd (i.e., 0.8% is 5% of 15%). Furthermore, the difference between practices equates to a difference in admission rate of 2.3% for participating patients . Taking the primary diagnosis mean admission rate of 14% (see table 2) before the start of the service for participating practices, the admission rate difference is equivalent to an average reduction in primary diagnosis admissions of 16% (95% ci=1 to 32%; c.f . This average reduction appears significant, whereas the difference of 0.8% between participating and control practices was not, because a further assumption was made that no participation means no effect, thus constraining the result to a narrower interval . Therefore, for an average practice with an admission rate of 43% for any diagnosis (see table 2), the effect of the service was a 5% drop in admissions with any diagnosis of copd (i.e., 2.3% is 5% of 43%). Furthermore, the difference between practices equates to a difference in admission rate of 7% for participating patients . Taking the mean admission rate of 42% for any diagnosis (see table 2) before the start of the service for participating practices, the admission rate difference was found to be equivalent to an average reduction in any diagnosis admissions of 16% (95% ci=6 to 26%). This study is the first to examine the impact of the healthy outlook service in england by examining the admission rates for all of the 661 participating practices where the participating copd patients were registered . Therefore, the effect of the healthy outlook service on the admission rates is expected to be dependent on the patient participation rate within each practice . Admission rates per practice are also dependent on the severity of copd of the patients registered in the practice as well as on other efforts made by the practice to address copd admissions . Furthermore, the risk to copd patients and therefore the risk of an emergency hospital admission will vary from year to year because of factors such as changes in levels of circulating viruses . While participating practices were clustered by commissioning organisation, usually primary care trusts, they present a wide geographical distribution illustrated in figure 4, such that it is unlikely that participating practices were climatically biased compared with the controls . A limitation of the eastern region public health observatory method for matching practices is that it does not take into account factors that might be linked to the take - up of the service, such as practices with a larger number of registered patients being more engaged in offering the service to patients . This would account for the significant difference in the number of copd patients registered in participating and control practices, with 18 more patients in participating practices (95% ci=11 to 25; p<0.001). The number of patients per practice could have been an additional matching criterion . Because it is already used as the denominator to measure the admission rate as outcome, the number of patients per practice was not used as a criterion for matching . A cohort study was carried out by steventon et al . For 1,413 copd patients matched to controls and enrolled in the healthy outlook service in 102 participating practices: they concluded that healthy outlook did not reduce admission rates but found lower mortality rates . A significant difference in their approach is in not having included unspecified and chronic bronchitis in the counts of copd hospital admissions . A possible explanation is that mild - to - moderate copd sufferers avoid hospitals following a telephone alert, whereas more severe sufferers will need emergency care in any case and despite the alert . Although anticipatory care interventions are often well received, copd disease management can yield significant direct health - care costs including the use of telemedicine . Although we have demonstrated a small but statistically significant reduction in hospital admissions for participating practices, it is unclear how the cost benefit of the healthy outlook can be quantified . Modelling the natural history of copd linked with health economics may provide a more precise cost benefit assessment . We found a 5% reduction in primary copd diagnosis hospital admission rates when comparing participating and control practices in the year following adoption of the healthy outlook service . While the difference was not statistically significant, this may be explained by the range of uptake of the service within the participating practices (11 and 81%). However, and possibly more importantly, we have demonstrated a small, statistically significant net reduction in hospital admission rates in patients with any primary or co - morbid diagnosis of copd . The importance of this finding emphasises the contribution of co - morbid copd on overall hospital admissions, which has a knock - on effect when planning health - care provision . Furthermore, when allowing for differences in participation rates (or exposure to the service), changes in admission rates for those with a primary or co - morbid diagnosis of copd are statistically significant and equivalent to a 16% drop in admissions . We have demonstrated that participation in the healthy outlook service of the met office reduces hospital admission rates for patients coded on discharge with a primary or co - morbid diagnosis of copd . This has major implications for patient well - being as well as for nhs resources . In the year after recruitment, the difference between participating and control practices was 0.8% for admissions with a primary diagnosis of copd . Therefore, for an average practice with a primary diagnosis admission rate of 15% (see table 2), the effect of the healthy outlook service was a 5% drop in admissions with a primary diagnosis of copd (i.e., 0.8% is 5% of 15%). Furthermore, the difference between practices equates to a difference in admission rate of 2.3% for participating patients . Taking the primary diagnosis mean admission rate of 14% (see table 2) before the start of the service for participating practices, the admission rate difference is equivalent to an average reduction in primary diagnosis admissions of 16% (95% ci=1 to 32%; c.f . This average reduction appears significant, whereas the difference of 0.8% between participating and control practices was not, because a further assumption was made that no participation means no effect, thus constraining the result to a narrower interval . Therefore, for an average practice with an admission rate of 43% for any diagnosis (see table 2), the effect of the service was a 5% drop in admissions with any diagnosis of copd (i.e., 2.3% is 5% of 43%). Furthermore, the difference between practices equates to a difference in admission rate of 7% for participating patients . Taking the mean admission rate of 42% for any diagnosis (see table 2) before the start of the service for participating practices, the admission rate difference was found to be equivalent to an average reduction in any diagnosis admissions of 16% (95% ci=6 to 26%). This study is the first to examine the impact of the healthy outlook service in england by examining the admission rates for all of the 661 participating practices where the participating copd patients were registered . Therefore, the effect of the healthy outlook service on the admission rates is expected to be dependent on the patient participation rate within each practice . Admission rates per practice are also dependent on the severity of copd of the patients registered in the practice as well as on other efforts made by the practice to address copd admissions . Furthermore, the risk to copd patients and therefore the risk of an emergency hospital admission will vary from year to year because of factors such as changes in levels of circulating viruses . While participating practices were clustered by commissioning organisation, usually primary care trusts, they present a wide geographical distribution illustrated in figure 4, such that it is unlikely that participating practices were climatically biased compared with the controls . A limitation of the eastern region public health observatory method for matching practices is that it does not take into account factors that might be linked to the take - up of the service, such as practices with a larger number of registered patients being more engaged in offering the service to patients . This would account for the significant difference in the number of copd patients registered in participating and control practices, with 18 more patients in participating practices (95% ci=11 to 25; p<0.001). Because it is already used as the denominator to measure the admission rate as outcome, the number of patients per practice was not used as a criterion for matching . A cohort study was carried out by steventon et al . For 1,413 copd patients matched to controls and enrolled in the healthy outlook service in 102 participating practices: they concluded that healthy outlook did not reduce admission rates but found lower mortality rates . A significant difference in their approach is in not having included unspecified and chronic bronchitis in the counts of copd hospital admissions . A possible explanation is that mild - to - moderate copd sufferers avoid hospitals following a telephone alert, whereas more severe sufferers will need emergency care in any case and despite the alert . Although anticipatory care interventions are often well received, copd disease management can yield significant direct health - care costs including the use of telemedicine . Although we have demonstrated a small but statistically significant reduction in hospital admissions for participating practices, it is unclear how the cost benefit of the healthy outlook can be quantified . Modelling the natural history of copd linked with health economics may provide a more precise cost benefit assessment . We found a 5% reduction in primary copd diagnosis hospital admission rates when comparing participating and control practices in the year following adoption of the healthy outlook service . While the difference was not statistically significant, this may be explained by the range of uptake of the service within the participating practices (11 and 81%). However, and possibly more importantly, we have demonstrated a small, statistically significant net reduction in hospital admission rates in patients with any primary or co - morbid diagnosis of copd . The importance of this finding emphasises the contribution of co - morbid copd on overall hospital admissions, which has a knock - on effect when planning health - care provision . Furthermore, when allowing for differences in participation rates (or exposure to the service), changes in admission rates for those with a primary or co - morbid diagnosis of copd are statistically significant and equivalent to a 16% drop in admissions . We have demonstrated that participation in the healthy outlook service of the met office reduces hospital admission rates for patients coded on discharge with a primary or co - morbid diagnosis of copd . This has major implications for patient well - being as well as for nhs resources.
Cardiovascular disease accounted for 30% of an estimated 58 million deaths globally from all causes in 2005 . It is important to recognise that a substantial proportion of these deaths (46%) occurred in individuals under 70 years of age, in their productive years of life; in fact, 79% of the disease burden attributed to cardiovascular disease is in this age group . Moreover, between 2006 and 2015, deaths due to non - communicable diseases (half of which are due to cardiovascular disease) are expected to increase by 17% . This projected increase is most prominent in asia due to a very large, ageing population, increasing exposure to a westernised diet, and rapidly increasing rates of diabetes mellitus . Early detection of patients at risk and early institution of effective treatment may help to ease the mortality and morbidity burden of cardiovascular disease . To achieve this, improved early diagnosis in patients with chest pain in or before the emergency department would reduce the burden on the hospitals . Furthermore, in an ageing population with improved outcome after myocardial infarction, the number of patients with an elevated risk for a second cardiovascular event is increasing but identification of patients at high risk of recurrent events remains difficult . Increasing survival following acute coronary events also leads to more patients living to develop heart failure (hf). Hf, the final common pathway of cardiac insults, is a serious syndrome due to inability of the heart to provide adequate circulation for the body s needs or only able to do so at increased cardiac filling pressures . It is the cardiovascular syndrome projected to increase most in the usa with the largest attendant loss of productivity and increase in direct medical cost in the next 20 years . Europe and asia show similar data [5, 6], with a rapid increase in disease burden on society . Evidence - based therapy of proven efficacy has been introduced over the last 25 years but mortality and readmission rates remain high . These vesicles are important in cell to cell communication in a variety of processes including coagulation, antigen presentation and tissue damage . Plasma extracellular vesicles can be easily isolated from frozen plasma or serum and contain protein, mirna and rna depending on source and stimulus . Extracellular vesicles that play a role in the initiation of thrombus formation are of major interest in cardiovascular disease pathogenesis, since thrombosis is the major underlying cause of cardiovascular events . In this process, tissue factor (tf), as an important initiator of thrombosis, was found in vesicles that are positive for the monocyte marker cd14 (fig . 1). In vitro studies using endothelial cells (ec) showed that these tf - containing vesicles do not only initiate thrombosis but also adhere to the ec that results in increased ec thrombogenicity via elevation of tf activity in the ec and decrease of tissue factor pathway inhibitor and thrombomodulin . This procoagulant activity, with monocyte - derived circulating tf as its main source, was associated with coronary artery disease and in stent thrombosis .fig . Subpopulations of extracellular vesicles (evs) in the plasma involved in coagulation are thought to have three origins: monocyte - derived evs (blue), platelet - derived evs (red) and endothelial cell - derived evs (green) as outlined in the enlarged ev 3-colour pie chart . For coagulation tf is the key initiator of the coagulation cascade and forms a complex with factor viia (fviia). The activated factor x (fxa) further cleaves prothrombin into thrombin that converts fibrinogen into fibrin to form clot . For fibrinolysis, the plasmin inhibitors serping1 and serpinf2 have been identified in evs and inhibit the breakdown of a fibrin via fibrinolysis . Abbreviations: ev, extracellular vesicle; tf, tissue factor; psgl-1, p - selectin glycoprotein ligand-1; ps, phosphatidylserine; p - sel, p - selectin; e - sel, e - selectin diagram showing an overview of the potential plasma extracellular vesicle procoagulant activity . Subpopulations of extracellular vesicles (evs) in the plasma involved in coagulation are thought to have three origins: monocyte - derived evs (blue), platelet - derived evs (red) and endothelial cell - derived evs (green) as outlined in the enlarged ev 3-colour pie chart . For coagulation tf is the key initiator of the coagulation cascade and forms a complex with factor viia (fviia). The activated factor x (fxa) further cleaves prothrombin into thrombin that converts fibrinogen into fibrin to form clot . For fibrinolysis, the plasmin inhibitors serping1 and serpinf2 have been identified in evs and inhibit the breakdown of a fibrin via fibrinolysis . Abbreviations: ev, extracellular vesicle; tf, tissue factor; psgl-1, p - selectin glycoprotein ligand-1; ps, phosphatidylserine; p - sel, p - selectin; e - sel, e - selectin if extracellular vesicle proteins are involved in cardiovascular events, then plasma vesicle protein content might be an important biomarker source for prognosis and prediction of cardiovascular events . Circulating mirnas are, at least partly, derived from extracellular vesicles and have been used in the diagnosis of acute coronary syndrome (acs,). Extracellular vesicle protein levels, however, have only recently been described to carry information in the presence of acs . Following a proteomics discovery approach, we demonstrated in a chest pain cohort of 471 patients that pigr, cystatin c (cst3), c5a and total vesicle protein concentration are associated with the presence of acs . Comparisons of this association between male and female patients reveal these markers to be highly associated with acs in males but not with acs in females . This striking difference is in agreement with an increasing body of evidence that women tend to have lower rates of coronary artery occlusions and have a more preserved left ventricular function . Women, however, also have a higher mortality and a worse prognosis following mi compared with men . The established difference in extracellular vesicle protein levels associated with acs identifies vesicles as an excellent biomarker source for gender - specific diagnosis and prognosis of acs . Furthermore, this might help in understanding the underlying gender differences in the pathophysiology of acs . The number of survivors of a first cardiovascular event is on the rise globally with a rapid increase in asia . These patients are at high risk for a second cardiovascular event even after adequate control of risk factors . The number and type of extracellular vesicles that are cd31/annexin v positive were associated with an increased risk for secondary events consisting of myocardial infarction, percutaneous coronary intervention (pci), coronary artery bypass graft (cabg), stroke and vascular mortality . For the first time, the protein content of all plasma extracellular vesicles and its association with secondary cardiovascular events were studied in a large clinical cohort . Comparing plasma extracellular vesicles of patients with a secondary cardiovascular event to gender- and age - matched patients without a second cardiovascular event these vesicle proteins were cystatin c, serping1, serpinf2 and cd14 and were validated in the smart cohort . This revealed that three of these vesicle proteins (cystatin c, serpinf2 and cd14) were associated with future cardiovascular events after correction of risk factors such as age, gender, smoking and egfr . For this, measurement of plasma extracellular vesicle protein levels has the potential to stratify patients at risk for secondary events on top of risk factors . There is much uncertainty over the relative contribution of platelets to the overall circulating extracellular vesicle population . Platelets are a key intermediary in the blood extracellular vesicle landscape because their function is readily and rapidly modified by platelet - directed medications . The ongoing stratifying risk with metabolomics and platelet reactivity testing in acute coronary syndrome (smart - acs) study, initiated in singapore, includes platelet reactivity testing of more than 1000 patients undergoing invasive management of acs, using impedance aggregometry (multiple electrode aggregometry, roche diagnostics, indianapolis, in) and follow up for secondary cardiovascular events . This will allow us to investigate how platelet - directed medications may potentially modify the content of the plasma extracellular vesicle population and its association with secondary events . The identification and validation of new biomarkers is highly dependent on the availability of patient cohorts with detailed phenotypic characterisation . Important ethnic differences have been described in the susceptibility, risk factors, and outcomes with cardiovascular disease . For example: indian men in the uk have an adjusted hazard ratio of 1.45 (ci: 1.29 to 1.63) for cardiovascular disease while chinese men in the uk have an adjusted hazard ratio of 0.51 (ci: 0.32 to 0.83) compared with caucasian men . Among southeast asian patients with hf in singapore, indians and malays have been shown to suffer worse outcomes than chinese although the reasons remain unclear . The adhere - asia pacific registry showed that compared with other multicentre registries in the us and europe, asian patients hospitalised with hf were younger and had more severe clinical features . For this, we initiated the (ongoing) biobanking efforts in both singapore and the netherlands (fig . 2). In 2008, the design of the atheroexpress collecting tissue, plasma and blood cells with a follow - up for secondary cardiovascular events was used to start up the singapore atheroexpress at the national university heart center singapore (nuhcs). This singapore biobank is collecting, besides atherectomy samples, also aortic punctures during cabg next to plasma and blood cells, and follows patients up for secondary cardiovascular events . Aortic punctures and plasma are now also collected at the umc utrecht in pegasus using the same design.fig . Red boxes indicate matched biobank studies, green boxes indicate work in progress of matching singapore and dutch biobank studies, numbers indicate patients included ~ june 2013 overview of ongoing cardiovascular biobanking in singapore and the netherlands . Red boxes indicate matched biobank studies, green boxes indicate work in progress of matching singapore and dutch biobank studies, numbers indicate patients included ~ june 2013 singapore initiated the collection of plasma of patients undergoing a pci with follow - up for secondary cardiovascular events . A year later, the umc utrecht mirror biobank of pci patients was initiated, a collective effort that now includes over 1000 patients a year . Work is in progress to match patients with chest pain in the emergency department of the meander hospital in amersfoort (minerva biobank cohort) with chest pain cohorts at nuhcs and singapore general hospital as well as the extensive hf cohort initiatives in singapore and asia with dutch heart failure cohorts . A key advantage of these mirrored cohorts is the implementation of common data and tissue collection and storage protocols across sites in both countries . This facilitates comparisons of biomarker profiles between dutch and singapore populations; such an effort is currently ongoing, with measurement of high sensitivity troponin t, high sensitivity c - reactive protein, von willebrand factor, myeloperoxidase and cystatin c in 1200 dutch and 1000 singaporean patients who have undergone elective coronary angiography . Plasma extracellular vesicle proteins are still in the early stage of study, mostly aiming to identify and to characterise the proteomes . Although relatively unexplored due to technical challenges, vesicle proteins hold promise as mass spectrometry - based proteomic technology is rapidly emerging for identification and quantification of extremely low abundant proteins in a sample . This has already resulted in identification of vesicle proteins for the diagnosis of acs and prognosis of secondary cardiovascular events in clinical cohorts . Isolation of plasma extracellular vesicle subpopulations is expected to lead to new and better cardiovascular vesicle protein biomarkers in a new sample set . This rapidly growing vesicle technology will hold, in combination with the maturation of the biobanks (inclusion of patients and secondary events) in the netherlands and asia, great promise for new diagnostic and prognostic biomarkers that take ethnicity and gender into account.
Kaposi sarcoma (ks) is characterized by multicentric neoplastic proliferation of vascular endothelial cells . Aids - related ks has a rapidly progressive course with early mucosal and systemic involvement . The incidence of aids - associated ks, especially the disseminated form, is very rare in india . Furthermore, the importance lies in early diagnosis and treatment which significantly affect the outcome . We are reporting the case for its rarity in india (probably the 18 case) and to highlight the importance of the early institution of highly active antiretroviral therapy (haart). A 34-year - old male presented with multiple asymptomatic pigmented raised lesions over the trunk, bilateral upper and lower limbs for last two months . He had history of fever and weight loss (> 10% total body weight) for the past six months . History of swelling of bilateral lower limbs, scrotum, and penis was present since one month . On examination, the patient had pallor, generalized lymphadenopathy, bilateral pitting pedal edema, and hepatosplenomegaly . Cutaneous examination revealed multiple discrete to grouped violaceous macules, papules, nodules and plaques measuring 0.2 cm 0.2 cm to 0.5 cm 3 cm over the trunk, bilateral upper and lower limbs [figure 1]. Cutaneous lesions over the lower abdomen and left thigh were coalescing to give a cobblestone appearance [figure 1]. Oral cavity showed two well - defined violaceous patches over the hard palate [figure 2]. On investigation, he had low hemoglobin (6.9 g / dl), deranged renal function parameters (urea - 69 mg / dl and creatinine - 1.9 mg / dl) and massive bilateral pleural effusion on chest x - ray . Serology for hiv-1 was positive by enzyme - linked immunosorbent assay and negative for hiv-2, and his cd4 count was 573/mm . Histopathology of skin biopsy specimen revealed proliferation of endothelial cells with formation of slit - like vascular spaces in the upper and middle dermis . Spindle - shaped endothelial cells were arranged in solid cords and fascicles admixed with variable lymphoplasmacytic infiltrate, hemorrhage, and lymphatic - like dilated spaces [figures 3 and 4]. Promontory sign, characterized by protruding of new vessels into the lumen of existing large vessel, was also demonstrated [figure 5]. On immunocytochemistry, a final diagnosis of aids - associated disseminated ks was made but unfortunately the patient died before starting haart because of recurrent massive pleural effusion . Multiple violaceous macules, papules, nodules, and plaques over the lower abdomen and bilateral thigh, some of which are coalescing to give cobblestone - like appearance two well - circumscribed violaceous patches present over the hard palate cord - like proliferation of spindle - shaped endothelial cells with areas of hemorrhage and variable lymphoplasmacytic infiltration in the dermis (h and e, 100) spindle - shaped endothelial cells arranged in fascicles in the dermis (h and e, 400) promontory sign - new blood vessels protruding into the lumen of existing blood vessel (arrow) (h and e, 400) cd31 positive proliferating endothelial cells (ihc, 100) human herpes virus-8 (hhv-8) is considered as the putative causative agent of ks . Co - infection of hhv-8 with hiv promotes the oncogenic capabilities of hhv-8, leading to the development of ks . The low incidence of ks in india is thought to be because of the low prevalence of the hhv-8 infection, with a prevalence of 4.7% reported from south india . Cutaneous lesions of classic ks start over the extremities as violaceous patches which gradually progress to form plaques and nodules . Progression is slow in the classic form, and mucosal and systemic involvement is rarely seen . The incidence of aids - associated ks is higher in homosexual males in comparison to females . Clinically, aids - related ks differs from the classical form because of its rapid clinical course and widespread dissemination that affect the mouth, lymph node, lungs, gastrointestinal tract and genitalia . Oral mucosal involvement can occur in all type of ks but more common in aids - associated ks . Common sites affected are hard palate and soft palate followed by gingiva, and dorsal tongue . The presence of oral lesion not only points towards undiagnosed hiv infection but also its association with the disseminated form of the disease which have potential diagnostic and therapeutic implications . In 50% patients of ks, oral mucosa is affected and in 20 - 25%, cases oral mucosa is the initial site of involvement . In indian patients, an involvement of both cutaneous and oral mucosa occurred in 61.53% cases while 38.46% had only cutaneous involvement . Our patient presented with two violaceous patches over hard palate which probably correlates with the rapidly progressive nature of the disease . In contrast to most of the malignancies, lymph node involvement in ks is not associated with worse prognosis and is of no special clinical significance . Lung involvement in ks occurs in 20% of the patients and is the most life - threatening form of the disease . Patients commonly present with progressive dyspnea, nonproductive cough, fever, chest pain and pleural effusion . Our patient had recurrent massive pleural effusion probably due to involvement of the lungs leading to the demise of the patient . From a dermatologist's point of view, ks should be kept in mind while dealing with an hiv - seropositive case with violaceous raised lesions, as the prognosis depends on the timely initiation of haart therapy . The closest mimics of ks are bacillary angiomatosis, pseudo - ks, lichen planus, malignant melanoma, pyogenic granuloma, and hemangioma . Immunohistochemical staining with hhv-8 clinches the diagnosis of ks, but in our case, we could not perform the test because of financial constraint . Aids - related ks responds well to haart therapy, and systemic chemotherapeutic drugs are indicated in nonresponsive patients or in patients with systemic involvement . Liposomal anthracyclines (doxorubicin and daunorubicin) are the united states food and drug administration approved first - line agent for treatment of ks . Due to its side - effect profile, other systemic treatment modalities include interferon- and combination chemotherapies with adriamycin, bleomycin plus vincristine, or vinblastine . In conclusion, our case highlights the rapidly progressive nature of aids - associated disseminated ks and importance of the early institution of haart . Early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi's sarcoma (ks) to achieve favorable prognosismucosal lesion can give a clue to the undiagnosed hiv . Early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi's sarcoma (ks) to achieve favorable prognosis mucosal lesion can give a clue to the undiagnosed hiv . Early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi's sarcoma (ks) to achieve favorable prognosismucosal lesion can give a clue to the undiagnosed hiv . Early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi's sarcoma (ks) to achieve favorable prognosis mucosal lesion can give a clue to the undiagnosed hiv . Early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi's sarcoma (ks) to achieve favorable prognosismucosal lesion can give a clue to the undiagnosed hiv . Early diagnosis and institution of antiretroviral therapy is crucial in aids - associated kaposi's sarcoma (ks) to achieve favorable prognosis mucosal lesion can give a clue to the undiagnosed hiv.
Based on review of our patient s medical record and current literature, we identified unique commonalities of clinical findings, imaging, and pathology for use in diagnosis and treatment planning . Samples of the patient s stored tissue were analyzed for immunohistochemical evidence of cytogenetic abnormalities . A 14-year - old caucasian female presented to the emergency department with a 2-week history of intermittent headaches associated with nausea and vomiting, becoming persistent and intensified in character . Initial magnetic resonance imaging (mri) of the brain and spine was limited by metal artifact from dental braces; however, leptomeningeal enhancement was noted diffusely throughout the brain and spinal cord with particular involvement of the basilar and suprasellar cisterns and distal lumbar nerve roots . The patient was admitted to the pediatric neurology service for evaluation of hydrocephalus and possible meningitis . Four days after admission, her condition deteriorated with development of visual acuity changes and bilateral sixth cranial nerve palsies . An external ventricular drain was placed, but continued progressive hydrocephalus necessitated diversion of cerebrospinal fluid (csf) via ventriculoperitoneal shunt . Cerebrospinal fluid studies, although repeatedly negative for malignant cells, consistently showed significant protein elevation . After removal of dental braces, mri demonstrated continued leptomeningeal enhancement and a new enhancing intramedullary lesion at c7-t2 measuring 4 17 mm (figure 1). Diffuse meningitic process and noted the focal signal changes were possibly the result of vascular thrombosis from infection or inflammation . During this initial hospitalization, extensive infectious and inflammatory workups were unrevealing, and the patient was discharged home . Postcontrast sagittal magnetic resonance imaging c - spine demonstrating dural enhancement (long arrows) and an enhancing intramedullary nodule at c7-t1 (short arrow). Eight months after presentation, new changes in mri included multifocal nonenhancing cystic lesions in the white matter . A month later, continued progressive leptomeningeal enhancement prompted repeat meningeal biopsies as well as brain tissue biopsies . Whole body bone scan and ct imaging of the chest, abdomen, and pelvis ruled out an occult neoplastic process with leptomeningeal dissemination . The biopsies revealed minimal chronic inflammation of the dura and unidentified atypical cells in the perivascular spaces determined to be of neuroepithelial origin by immunohistochemical staining . There was no infiltration of these cells into the vessels nor was there evidence of infarct . Systemic steroid therapy was considered and, prior to initiation, the patient was referred to pediatric neurooncology for consultation . Magnetic resonance imaging revealed increased dural thickening and enhancement with a new 1-cm mass - like lesion along the left cavernous sinus . There were also an increased number and size of cysts inferiorly and bilaterally in the brain, thought to represent abnormal dilatation of the perivascular spaces (figure 2), consistent with previous biopsy findings . Despite emergence of new findings and progression of dural thickening, the spinal lesion at the cervicothoracic junction was stable . A repeat biopsy was recommended . Our patient s progression of perivascular cystic development from initial presentation (a) to 3 months later (b). These final biopsies demonstrated cells with moderate pleomorphism and fine cytoplasmic processes, positive for glial fibrillary acid protein (gfap) and synaptophysin, and negative for isocitrate dehydrogenase 1 (r123h mutation - specific antibody). This was now interpreted to be a high - grade glioneuronal tumor due to a high mitotic rate and p53 positivity . Magnetic resonance imaging after 6 weeks of temozolomide and craniospinal irradiation revealed a significant decrease in contrast enhancement throughout the brain and spinal cord . The patient completed 6 weeks of craniospinal irradiation and oral temozolomide, followed by maintenance therapy with temozolomide 5 days each month for 1 year . There was complete resolution of symptoms and all magnetic resonance mri findings with the exception of persistence of the spinal nodule, which no longer enhanced with contrast administration . At the time of manuscript preparation armao et al described a case of an 8-year - old boy who presented with intermittent headaches . Although he had no initial hydrocephalus, over the following 6 months he had fluctuating neurologic signs and symptoms including papilledema, diplopia, ataxia, and upper extremity weakness . Communicating hydrocephalus developed over the first year, and over a 5-year period, the patient had progressive hydrocephalus, seizures, and intermittent encephalopathy . He also underwent csf studies revealing elevated protein but no malignant cells . On mr imaging, he had diffuse thickening and enhancement of the subarachnoid space and leptomeninges of the brain and spinal cord, more concentrated in the basal cisterns, sylvian fissures, and interhemispheric fissures . He had cystic lesions of the basal cisterns, basal ganglia, medulla, and cerebellar white matter . All cystic lesions noted were reported to have similar signal intensity to csf . With the development of leptomeningeal enhancement and the appearance of the cystic lesions, he was not treated for malignancy and subsequently died secondary to necrotizing pneumonia and fulminant sepsis . Postmortem biopsy showed tumor cell infiltrates in the perivascular spaces of the basal ganglia and cerebellum . The tumor cells were positive for s100 and leu-7 markers and negative for gfap human melanoma black 45, keratin, and cd45 . Although the tumor was negative for gfap (a glial cell marker), leu-7 is involved in myelination and is often seen in oligodendrogliomas . The author noted that this type of tumor usually has a primary intra - axial mass, which this case lacked . However, like our patient, he had an 8-mm oval - shaped, enhancing nodule at c6 to c7 . Gardiman et al presented 4 relatively similar cases: all childhood / adolescence presenting with neurologic manifestations, communicating hydrocephalus with spinal fluid high in protein and negative for malignant cells, diffuse leptomeningeal enhancement on imaging with cystic lesions thought to represent perivascular infiltration of tumor cells, and none of which had a primary tumor identified . Biopsies obtained in all 4 cases identified tumor cells with the morphologic appearance of glial differentiation but a significant number of nuclei were immunopositive for neu - n, and tumor cells were diffusely positive for synaptophysin . Only case 3 was found to have a 1p deletion on initial fluorescence in situ hybridization testing; however, on his second biopsy, there was a 50% mib-1 index and further progression to 1p19q codeletion . Case 3 received irradiation due to disease progression with development of tumor 2 years after finishing 12 months of temozolamide . None were disease free after treatment but progressed with a slow indolent course while the child in case 2 subsequently died from disease progression . In 2012, gardiman et al published an update on case 4 . The patient presented 22 months after the initial presentation and biopsy, with new seizures and personality changes . Magnetic resonance imaging at this time showed multiple intraparenchymal cysts and thick leptomeningeal enhancement with aggregation of tumor deposits in the dural sac . His first biopsy at initial presentation did not clearly provide adequate information for classification of the tumor . It revealed cells with copositivity for both neurocytes and oligodendrocytes, high mitotic index, and no cytogenetic abnormalities were detected . With this second presentation, a repeat biopsy showed a low mitotic index and low mib-1 in addition to the aforementioned features . The final diagnosis was determined to be a low - grade tumor consistent with diffuse leptomeningeal glioneuronal tumor and he was started on chemotherapy as per a low - grade protocol, which included vincristine and carboplatin . Due to severe neurotoxicity and prolonged myelosuppression, his therapy was initially switched to temozolamide alone . Then, for seizure and behavioral control, as well as the potential augmentation of glial differentiation, daily valproic acid was started and the temozolamide was discontinued (reason for its discontinuation not specified). Three months after initiating the valproic acid, a total of 67 months since initial presentation, the patient s leptomeningeal involvement and neurologic symptoms (including seizures and behavioral changes) were steadily improving . Since the description of diffuse leptomeningeal glioneuronal tumor, schniederjan et al reported 9 additional cases that they termed diffuse leptomeningeal neuroepithelial tumor . The ages of the children in this series ranged from 2 to 7 years old . The clinicopathologic presentation of their patients is almost identical to the above - described cases, including that of our patient . Of the 9 patients, 8 had cytogenetic testing for 1p/19q deletions . Of those tested, 6 demonstrated the 1p deletion, and 2 of those 6 also had the 19q codeletion . Of those with only the 1p deletion, 1 did not have sufficient tissue adherence for successful 19q fluorescence in situ hybridization testing . Of the 9 cases tested for deletions, 8 were also tested for isocitrate dehydrogenase 1 mutation and found to be negative . Overall, 2 patients were treated with temozolamide alone, 2 with temozolamide and other chemotherapy agents, 1 with vincristine and carboplatin, 1 with radiation alone, and 1 patient was treated with chemotherapy (drug not specified) and radiation therapy . At the time of their publication, 2 patients were newly diagnosed and had not yet started treatment . Abbreviations: lm, leptomeningeal; ip, intraparenchymal; dlgnt, diffuse leptomeningeal glioneuronal tumor; dlo, diffuse leptomeningeal oligodendrogliomatosis; dlnet, diffuse leptomeningeal neuroepithelial tumor; gfap, glial fibrillary acid protein; csf, cerebral spinal fluid; flair, fluid - attenuated inversion recovery; yo, years old; f, female; m, male . Reported as 1 cm mass in left cavernous sinus . Left frontal intraventricular lesion with perivascular pseudorosettes versus pseudopapillary structure . Internal auditory canals . Our patient s cysts were nonenhancing; armao patient s cyst enhanced like csf; all 4 gardiman patients cysts were hyperintense on t2 and isohypointense on t1 and flair . Gfap negative, leu-7 positive; leu-7 is associated with myelin and is present in a majority of oligodendrogliomas . Biopsy at initial presentation high mitotic index; biopsy at second presentation low mitotic index and <3% mib-1 . Dlo usually has a primary intra - axial mass this case did not . It has been believed that central nervous system neoplasms classically disseminate caudally from the origin of the primary tumor . However, of the 15 above - mentioned cases, 7 have a noted tumor or nodules found in the spinal cord with diffuse leptomeningeal enhancement and subsequent cystic changes in the brain . This suggests this tumor can indeed be a primary spinal cord neoplasm demonstrating cranial dissemination . Additionally, diffuse leptomeningeal enhancement is usually assumed to be evidence of dissemination associated with more progressive disease . The patients described with this unique tumor seem to present with hydrocephalus and diffuse leptomeningeal enhancement and do not usually present with cystic changes . They later develop cystic changes in the leptomeninges and parenchyma, which continue to increase in number and size with progression of the disease . This suggests the leptomeningeal enhancement is not necessarily a marker of advanced disease in these patients, but development and progression of cystic changes may be . Additionally, as the cysts appear to be noncomplex in nature (i.e., without components of protein or blood), these cystic changes are likely perivascular spaces enlarging with collections of extracellular fluid developing secondary to progression of the leptomeningeal disease altering normal drainage pathways . Mixed glioneuronal tumors and gangliogliomas are defined as consisting of 2 distinct cell populations, one population of neuronal cells mixed with another distinct population of glial cells . The glioneuronal tumors discussed in this article are composed of a singular cell population staining positively for markers of both glial and neuronal cell components . Therefore, histological staining for multiple markers of both types of cells in all samples collected can lead to earlier diagnosis . In our patient s case, not all biopsy samples included tumor cells, and different samples revealed varying levels of differentiation and of aggressive features . Gardiman case 4 also demonstrated differing levels of mitotic index at initial and later biopsies, as well as varying degrees of aggressive histological signs . Obtaining early and repeated biopsies, each with multiple sample sites, is more likely to provide the most important information leading to an earlier diagnosis of malignancy in patients with diffuse leptomeningeal enhancement and elevated csf protein without evidence of a primary tumor or malignant cells in the spinal fluid . Additionally, testing these multiple samples for both glial and neuronal cell markers, p53, olig-2, isocitrate dehydrogenase 1, and 1p/19q deletion can help with diagnosis, as well as give more information of prognosis and treatment options . However, it has been found in cells of glioneuronal tumors exhibiting both glial and neuronal characteristics . This can suggest a common progenitor cell type between glial and neuronal cells, giving support to the theory that these glioneuronal tumors are neoplasms of a common precursor cell for both glial and neuronal cell types . Somatic mutations of the idh1 gene have been associated with several forms of cancer, including astrocytomas, oligodendrogliomas, and hematopoetic dysplasias . As it is seen in over 70% of grade ii to iv gliomas and is not demonstrated in diffuse leptomeningeal glioneuronal tumors, isocitrate dehydrogenase 1 may be a useful marker in testing to differentiate diffuse leptomeningeal glioneuronal tumors from higher grade glial neoplasms . Our patient and the 9 schniederjan patients all tested negative for isocitrate dehydrogenase 1 (the others did not have isocitrate dehydrogenase 1 status reported). Chromosomal analysis for 1p19q codeletion, or even a singular deletion of either 1p or 19q, can be useful in determining tumor aggressiveness and likely response to chemotherapy, especially to temozolomide . This codeletion has been seen in both neurocytomas and adult oligodendrogliomas; and its being seen in both tumor types can also support the idea of a common progenitor cell being the neoplastic culprit in diffuse leptomeningeal glioneuronal tumors . This codeletion has demonstrated correlation with more aggressive disease in extraventricular neurocytomas and can be useful in assessment of potentially aggressive behavior of other tumor types, including diffuse leptomeningeal glioneuronal tumors . On the other hand, it has also been highly correlative with increased responsiveness to systemic chemotherapy, especially with temozolomide, with and without adjunctive radiotherapy, as well as disease prognosis . It is clear from the reported cases mentioned herein that this neoplasm is, in fact, treatable . Stable disease has been achieved with chemotherapy alone, as well as with adjunctive irradiation . As evidenced by our patient s case, therefore, treatment should absolutely be initiated, although the most effective treatment has not yet been identified . Our patient s remarkable response to her treatment regimen could have been due to the combined temozolamide and radiation therapy, which was allowable given her age . Her p53 positivity was also likely significant in her response to therapy, which can have represented a more aggressive tumor with higher sensitivity to the treatment modalities used . The seemingly significant response to initiation of valproic acid in gardiman case 4, both with improvement in neurological symptoms and radiographic findings, can indicate valproic acid lends itself to successful adjunctive therapy in cases with seizures and behavioral changes, and possibly even in those without . The majority of the other cases mentioned did not receive radiotherapy in addition to temozolamide, likely due to patient age, and subsequently had reportedly stable disease . With our patient s quick and steady response to her combined therapy regimen, it may be advisable to use combined treatment with systemic chemotherapy and craniospinal irradiation from the beginning more aggressive treatment for a potentially aggressive tumor . However, the risks involved depend on the individual patient and should still be tailored accordingly . Although temozolomide is not the only option for chemotherapy, it certainly has its advantages for the patient oral administration, less patient toxicity, and it seems to work well for this neoplasm . Suggestions for future studies, which can provide information leading to more successful therapy, include comparing chemotherapy with temozolomide alone versus chemotherapy with other agents, chemotherapy alone versus chemotherapy in conjunction with craniospinal irradiation, and the efficacy of valproic acid as an adjunctive therapy for patients with and without symptoms including seizures and/or behavioral changes . Additionally, and even more importantly, evaluation of the molecular characteristics of the disease will help targeted therapy . It has been believed that central nervous system neoplasms classically disseminate caudally from the origin of the primary tumor . However, of the 15 above - mentioned cases, 7 have a noted tumor or nodules found in the spinal cord with diffuse leptomeningeal enhancement and subsequent cystic changes in the brain . This suggests this tumor can indeed be a primary spinal cord neoplasm demonstrating cranial dissemination . Additionally, diffuse leptomeningeal enhancement is usually assumed to be evidence of dissemination associated with more progressive disease . The patients described with this unique tumor seem to present with hydrocephalus and diffuse leptomeningeal enhancement and do not usually present with cystic changes . They later develop cystic changes in the leptomeninges and parenchyma, which continue to increase in number and size with progression of the disease . This suggests the leptomeningeal enhancement is not necessarily a marker of advanced disease in these patients, but development and progression of cystic changes may be . Additionally, as the cysts appear to be noncomplex in nature (i.e., without components of protein or blood), these cystic changes are likely perivascular spaces enlarging with collections of extracellular fluid developing secondary to progression of the leptomeningeal disease altering normal drainage pathways . Mixed glioneuronal tumors and gangliogliomas are defined as consisting of 2 distinct cell populations, one population of neuronal cells mixed with another distinct population of glial cells . The glioneuronal tumors discussed in this article are composed of a singular cell population staining positively for markers of both glial and neuronal cell components . Therefore, histological staining for multiple markers of both types of cells in all samples collected can lead to earlier diagnosis . In our patient s case, not all biopsy samples included tumor cells, and different samples revealed varying levels of differentiation and of aggressive features . Gardiman case 4 also demonstrated differing levels of mitotic index at initial and later biopsies, as well as varying degrees of aggressive histological signs . Obtaining early and repeated biopsies, each with multiple sample sites, is more likely to provide the most important information leading to an earlier diagnosis of malignancy in patients with diffuse leptomeningeal enhancement and elevated csf protein without evidence of a primary tumor or malignant cells in the spinal fluid . Additionally, testing these multiple samples for both glial and neuronal cell markers, p53, olig-2, isocitrate dehydrogenase 1, and 1p/19q deletion can help with diagnosis, as well as give more information of prognosis and treatment options . However, it has been found in cells of glioneuronal tumors exhibiting both glial and neuronal characteristics . This can suggest a common progenitor cell type between glial and neuronal cells, giving support to the theory that these glioneuronal tumors are neoplasms of a common precursor cell for both glial and neuronal cell types . Somatic mutations of the idh1 gene have been associated with several forms of cancer, including astrocytomas, oligodendrogliomas, and hematopoetic dysplasias . As it is seen in over 70% of grade ii to iv gliomas and is not demonstrated in diffuse leptomeningeal glioneuronal tumors, isocitrate dehydrogenase 1 may be a useful marker in testing to differentiate diffuse leptomeningeal glioneuronal tumors from higher grade glial neoplasms . Our patient and the 9 schniederjan patients all tested negative for isocitrate dehydrogenase 1 (the others did not have isocitrate dehydrogenase 1 status reported). Chromosomal analysis for 1p19q codeletion, or even a singular deletion of either 1p or 19q, can be useful in determining tumor aggressiveness and likely response to chemotherapy, especially to temozolomide . This codeletion has been seen in both neurocytomas and adult oligodendrogliomas; and its being seen in both tumor types can also support the idea of a common progenitor cell being the neoplastic culprit in diffuse leptomeningeal glioneuronal tumors . This codeletion has demonstrated correlation with more aggressive disease in extraventricular neurocytomas and can be useful in assessment of potentially aggressive behavior of other tumor types, including diffuse leptomeningeal glioneuronal tumors . On the other hand, it has also been highly correlative with increased responsiveness to systemic chemotherapy, especially with temozolomide, with and without adjunctive radiotherapy, as well as disease prognosis . It is clear from the reported cases mentioned herein that this neoplasm is, in fact, treatable . Stable disease has been achieved with chemotherapy alone, as well as with adjunctive irradiation . As evidenced by our patient s case, therefore, treatment should absolutely be initiated, although the most effective treatment has not yet been identified . Our patient s remarkable response to her treatment regimen could have been due to the combined temozolamide and radiation therapy, which was allowable given her age . Her p53 positivity was also likely significant in her response to therapy, which can have represented a more aggressive tumor with higher sensitivity to the treatment modalities used . The seemingly significant response to initiation of valproic acid in gardiman case 4, both with improvement in neurological symptoms and radiographic findings, can indicate valproic acid lends itself to successful adjunctive therapy in cases with seizures and behavioral changes, and possibly even in those without . The majority of the other cases mentioned did not receive radiotherapy in addition to temozolamide, likely due to patient age, and subsequently had reportedly stable disease . With our patient s quick and steady response to her combined therapy regimen, it may be advisable to use combined treatment with systemic chemotherapy and craniospinal irradiation from the beginning more aggressive treatment for a potentially aggressive tumor . However, the risks involved depend on the individual patient and should still be tailored accordingly . Although temozolomide is not the only option for chemotherapy, it certainly has its advantages for the patient oral administration, less patient toxicity, and it seems to work well for this neoplasm . Suggestions for future studies, which can provide information leading to more successful therapy, include comparing chemotherapy with temozolomide alone versus chemotherapy with other agents, chemotherapy alone versus chemotherapy in conjunction with craniospinal irradiation, and the efficacy of valproic acid as an adjunctive therapy for patients with and without symptoms including seizures and/or behavioral changes . Additionally, and even more importantly, evaluation of the molecular characteristics of the disease will help targeted therapy . In conclusion, when patients present with sequelae of nonspecific neurologic signs and symptoms, hydrocephalus, csf studies significant only for elevated protein without evidence of malignant cells, and diffuse leptomeningeal enhancement on radiographic images, the differential diagnosis should include diffuse leptomeningeal glioneuronal tumor (also described as diffuse leptomeningeal neuroepithelial tumor). Additionally, if cystic changes are seen with the above - mentioned findings, it should prompt aggressive workup for this tumor in addition to the infectious workup usually employed . Olig-2 may be a marker implying a common progenitor cell for neurocytes and glial cells in tumors . Although p53 and 1p19q codeletions represent more aggressive tumor activity, 1p19q codeletions have shown significantly increased responsiveness to chemotherapy, especially temozolomide . When this diagnosis is in the differential, it is advisable to obtain earlier (and possibly repeated) biopsies from multiple sites, and these tissue samples should be tested for the following factors: multiple glial cell markers, multiple neuronal cell markers, p53, olig-2, isocitrate dehydrogenase 1, and chromosomal deletions (especially of 1p and 19q). Obtaining this information can lead to earlier diagnosis and treatment of diffuse leptomeningeal glioneuronal tumors in patients who present with hydrocephalus and diffuse leptomeningeal enhancement without evidence of a primary tumor . Temazolomide seems to be effective and is especially recommended for patients with markers of more aggressive tumor activity . Both medications should be considered when appropriate and investigated in more depth in future studies . This is a seemingly low - grade tumor and collective efforts should be undertaken to develop molecular characterization of this entity in hopes of developing targeted therapy.
Triangular fibrocartilage complex (tfcc) injury is one of the most common causes of the ulnar sided wrist pain and distal radioulnar joint (druj) disorder . The ulnar side of the tfcc is divided into three components based on histologic and functional studies: the distal hammock like structure, the proximal ligamentous component and the ulnar collateral ligament.1 the proximal ligament, composed of the anterior and posterior distal radioulnar ligaments, converges on the foveal site of the ulnar head, contributing as an important stabilizing structure of the druj . Disruption of the proximal ligament from its foveal insertion can lead to druj instability accompanied by ulnar - sided pain, weakness, snapping and limited forearm rotation.23 several authors have performed open repair of tfcc foveal tear as a mainstay of treatment.45 although they reported favorable clinical results, arthroscopic assisted repair has been recently advocated as an alternative because it is less invasive.6 this study investigates the clinical results in patients with tfcc foveal tears who underwent arthroscopic assisted repair and describe a modified technique (a knotless suture without bone tunnel) of arthroscopic reattachment of avulsed tfcc foveal tears . 12 patients who underwent arthroscopic foveal repair between march 2011 and december 2013 were included in this retrospective study . Tfcc foveal tear was diagnosed based on: (1) positive foveal sign (foveal tenderness) and presence of instability on druj stress test78 compared with the contralateral side (2) a magnetic resonance image (mri) and (3) a positive hook test on arthroscopic examination . While the patient's distal radius was grasped by a one hand, the distal ulna was passively translated in the dorsal and palmar directions . The test was repeated with the forearm in supination and pronation positions and the results were compared with the contralateral normal side . Four levels of instability severity was classified depending on the degree of translation, presence or absence of pain and the resistance at the end point; normal as grade 0, increased laxity but a firm end point and without pain as grade 1, increased translation with a soft end point accompanied by pain or apprehension as grade 2, and druj subluxation that occurred during the forearm rotation or with limited rotation was classified as grade 3.9 arthroscopic repair was primarily done in patients with grade 2 dynamic instability . The exclusion criteria were: (1) arthritic change of druj on radiographs, (2) evidence of ulnocarpal impingement, or (3) a malunion state following distal radioulnar fracture . There were 11 males and 1 female with a mean age of 24.7 years (range 1734 years). All patients had a history of wrist trauma and had conservative treatment for the ulnar side wrist pain for 6 months or longer . Mri revealed a high signal intensity area just distal to the ulnar fovea on t2-weighted coronal images in 9 patients, indicating a disruption of tfcc foveal insertion . Arthroscopy showed a complete peripheral tfcc tear involving both the distal and proximal components (class 2) in 4 patients and an isolated proximal foveal tear (class 3) in 8 patients according to atzei et al . Classification.1 the mean time delay from injury to operation was 12.2 months (range 436 months), and the mean followup period was 19 months (range 1425 months). At the final followup, we measured range of motion (rom) and grip strength, investigated druj instability based on the stress test and assessed pain and function using the visual analog scale (vas) for pain, patient rated wrist evaluation (prwe) score and the disabilities of the arm, shoulder and hand (dash) questionnaires . Arthroscopic view showing the hook test (a) the probe is inserted through the 6r portal, and traction is applied to the ulnar prestyloid recess . (b) the test is positive when the triangular fibrocartilage complex can be folded and pulled upward and radially by the probe we performed pull out suture repair through the bone tunnel in the first 6 patients, and knotless suture anchor repair in remaining 6 patients . All patients underwent a brachial plexus block with a tourniquet applied to the proximal arm . The wrist was fixed on the traction tower (conmed linvatec, largo, fl, usa) with 1015 pounds of traction . After systemic examination of the radiocarpal joint, the trampoline test10 and the hook test11 were performed to confirm the diagnosis of a tfcc foveal avulsion . A 2-cm longitudinal skin incision was made between the flexor carpi ulnaris, and extensor carpi ulnaris tendons . With the wrist in semisupinated position, the ulnar foveal site was approached beneath the tfcc through the incision of the volar capsule . We removed the fibrous tissue and scraped the foveal portion of the ulnar head using a curet to expose the subchondral bone . A suture hook (linvatec, largo, fl, usa) loaded with a 10 polydioxanone ii (pds ii; ethicon, livingston, united kingdom) surgical suture was penetrated through the peripheral edge of the tfcc in a bottom - to - top fashion with the scope in the 34 portal . The end of the passed pds ii suture was retrieved through the 45 portal using a grasper . A nylon surgical suture was passed through the tfcc at an interval from the first suture and was used as a shuttle relay to pass the pds ii suture . A horizontal mattress loop was established by pulling the nylon suture through the incised wound [figure 2]. Two tunnels were created from the ulnar neck to the fovea using a kirschner wire and each pds ii suture end was delivered to the proximal entrance of the tunnel . Both suture ends were pulled to reduce the tfcc to the fovea and fixed tightly by knotting on the ulnar neck portion [figure 3]. The hook test was carried out again to confirm the restoration of peripheral tfcc stability . Arthroscopic view showing a horizontal mattress loop the suture is tied under arthroscopic vision, and triangular fibrocartilage complex is pushed forcefully against the fovea of the distal ulna peroperative photograph showing transosseous pull - out suture repair . Both suture ends are pulled out through the bone tunnel to reduce the triangular fibrocartilage complex to the fovea and fixed tightly by knotting on the ulnar neck portion the same arthroscopic procedure was performed as described above . Approximately 1.5-cm transverse skin incision was made and the ulnar foveal site was approached through the volar capsule . A mattress suture loop using a 20 fiberwire (arthrex, naples, fl, usa) was established in the same manner as the transosseous pull - out technique . Instead of making a bone tunnel, a 2.5-mm knotless suture anchor (pushlock; arthrex, naples, fl, usa) was used to fix the tfcc foveal site avulsion [figure 4]. After a hole was drilled at the foveal insertion site, both ends of suture strands these were placed into the drill hole with tension and securely fixed by tapping the button on the proximal end of the driver handle to impact the anchor body portion into the bone [figures 5 and 6]. Both ends of suture strands are passed through the eyelet of the 2.5 mm knotless suture anchor (pushlock) without bone tunnel diagrammatic representation of the triangular fibrocartilage complex foveal repair by using knotless suture anchor . (a) through the 1.5 cm transverse skin incision on the volar capsule, both ends of sling suture strands are passed through the eyelet of the knotless suture anchor . (b) the anchor is placed into the drill hole with tension, and securely fixed into the bone representative example of the triangular fibrocartilage complex foveal avulsion and postoperative magnetic resonance images of a 20-year - old man . (a) coronal t2 fat - suppressed proton density - weighted image showing the triangular fibrocartilage complex foveal avulsion . (b) three months postoperative coronal image showing a knotless suture anchor and recovered continuity of triangular fibrocartilage complex on the ulnar fovea . (c) postoperative axial t1-weighted image showing proper location of the knotless suture anchor on the ulnar fovea long arm cast immobilization with the forearm in the neutral rotation was maintained for 4 weeks . It was substituted by a long arm thermoplastic brace and wrist motion exercise was initiated . The patients were instructed to perform active assisted wrist mobilization and supination / pronation of forearm until the full motion was restored . Chicago, il, usa) program was used for the statistical analysis of our data . We used a wilcoxon signed rank test to compare the preoperative and postoperative rom, grip strength, and score changes . We performed pull out suture repair through the bone tunnel in the first 6 patients, and knotless suture anchor repair in remaining 6 patients . All patients underwent a brachial plexus block with a tourniquet applied to the proximal arm . The wrist was fixed on the traction tower (conmed linvatec, largo, fl, usa) with 1015 pounds of traction . After systemic examination of the radiocarpal joint, the trampoline test10 and the hook test11 were performed to confirm the diagnosis of a tfcc foveal avulsion . A 2-cm longitudinal skin incision was made between the flexor carpi ulnaris, and extensor carpi ulnaris tendons . With the wrist in semisupinated position, the ulnar foveal site was approached beneath the tfcc through the incision of the volar capsule . We removed the fibrous tissue and scraped the foveal portion of the ulnar head using a curet to expose the subchondral bone . A suture hook (linvatec, largo, fl, usa) loaded with a 10 polydioxanone ii (pds ii; ethicon, livingston, united kingdom) surgical suture was penetrated through the peripheral edge of the tfcc in a bottom - to - top fashion with the scope in the 34 portal . The end of the passed pds ii suture was retrieved through the 45 portal using a grasper . A nylon surgical suture was passed through the tfcc at an interval from the first suture and was used as a shuttle relay to pass the pds ii suture . A horizontal mattress loop was established by pulling the nylon suture through the incised wound [figure 2]. Two tunnels were created from the ulnar neck to the fovea using a kirschner wire and each pds ii suture end was delivered to the proximal entrance of the tunnel . Both suture ends were pulled to reduce the tfcc to the fovea and fixed tightly by knotting on the ulnar neck portion [figure 3]. The hook test was carried out again to confirm the restoration of peripheral tfcc stability . Arthroscopic view showing a horizontal mattress loop the suture is tied under arthroscopic vision, and triangular fibrocartilage complex is pushed forcefully against the fovea of the distal ulna peroperative photograph showing transosseous pull - out suture repair . Both suture ends are pulled out through the bone tunnel to reduce the triangular fibrocartilage complex to the fovea and fixed tightly by knotting on the ulnar neck portion approximately 1.5-cm transverse skin incision was made and the ulnar foveal site was approached through the volar capsule . A mattress suture loop using a 20 fiberwire (arthrex, naples, fl, usa) was established in the same manner as the transosseous pull - out technique . Instead of making a bone tunnel, a 2.5-mm knotless suture anchor (pushlock; arthrex, naples, fl, usa) was used to fix the tfcc foveal site avulsion [figure 4]. After a hole was drilled at the foveal insertion site, both ends of suture strands these were placed into the drill hole with tension and securely fixed by tapping the button on the proximal end of the driver handle to impact the anchor body portion into the bone [figures 5 and 6]. Both ends of suture strands are passed through the eyelet of the 2.5 mm knotless suture anchor (pushlock) without bone tunnel diagrammatic representation of the triangular fibrocartilage complex foveal repair by using knotless suture anchor . (a) through the 1.5 cm transverse skin incision on the volar capsule, both ends of sling suture strands are passed through the eyelet of the knotless suture anchor . (b) the anchor is placed into the drill hole with tension, and securely fixed into the bone representative example of the triangular fibrocartilage complex foveal avulsion and postoperative magnetic resonance images of a 20-year - old man . (a) coronal t2 fat - suppressed proton density - weighted image showing the triangular fibrocartilage complex foveal avulsion . (b) three months postoperative coronal image showing a knotless suture anchor and recovered continuity of triangular fibrocartilage complex on the ulnar fovea . (c) postoperative axial t1-weighted image showing proper location of the knotless suture anchor on the ulnar fovea long arm cast immobilization with the forearm in the neutral rotation was maintained for 4 weeks . It was substituted by a long arm thermoplastic brace and wrist motion exercise was initiated . The patients were instructed to perform active assisted wrist mobilization and supination / pronation of forearm until the full motion was restored ., chicago, il, usa) program was used for the statistical analysis of our data . We used a wilcoxon signed rank test to compare the preoperative and postoperative rom, grip strength, and score changes . The mean postoperative arc of flexion extension, radial - ulnar deviation, and pronation - supination were 147 (range 115180), 55 (range 3580), and 166 (range 160180) respectively at the final followup . The flexion extension (p = 0.088) and radial - ulnar deviation (p = 0.228) did not change significantly, while the range of pronation supination of the forearm increased significantly (p = 0.04). The mean grip strength increased from 54.9 26.0% of the contralateral side preoperatively to 72.8 24.7% postoperatively . However, it did not reach statistical significance (p = 0.051). Before the operation, all patients had grade 2 druj instability . At the final followup, druj stress test showed improved stability in all patients (grade 0 in 5 patients and grade 1 in 7 patients). Based on the vas for pain, there was a statistically significant improvement in postoperative pain compared with preoperative pain (from 5.3 to 1.7) (p = 0.003). Four patients showed no pain, and 6 patients complained of mild pain (vas 12). Mean prwe score for pain improved significantly from 33.3 (range 1944) preoperatively to 20.7 (range 636) postoperatively (p = 0.003), and the mean prwe for function also improved significantly from 58.7 (range 2285) to 30.2 (884) (p = 0.007). Dash questionnaire scores showed a significant improvement from 48.89 (range 21.5573.28) preoperatively to 24.6 (range 9.1760) postoperatively (p = 0.005). That patient was able to return to a previous job with limited activity due to moderate pain and restricted range of supination . While druj stability can be preserved when there is an isolated tear of the distal capsular portion of the tfcc, disruption of the proximal ligament from the foveal insertion causes instability of the druj . When the foveal disruption is overlooked, the results may be unsatisfactory even after arthroscopic capsular repair.12 the diagnosis of tfcc injury is usually made based on the arthroscopy and mri findings however, detection of foveal disruption of the tfcc is challenging, especially when the peripheral capsular portion is intact . Consequently, we made the diagnosis mainly on the results of a clinical stress test.9 on the basis of the stress test results, we suggest that grade 2 dynamic druj instability is the most appropriate indication for the arthroscopic foveal repair . Grade 1 represents laxity, for which surgical repair is not necessary and grade 3 may require open repair or reconstruction of the distal radioulnar ligament to reduce the gross subluxation . Mri may be helpful to diagnose the tfcc foveal tear when it shows high signal intensity with discontinuity of ligamentous fibers to the foveal area; however, its value is limited because of inconsistent findings.13 in our study, several cases failed to show definite lesions on the mri . A positive hook test during an arthroscopic operation has been suggested as a reliable sign of foveal disruption.1 because the results are based on a subjective decision, we experienced many cases in which the test was inconclusive . Some authors used druj arthroscopy to observe the foveal insertion directly.141516 however, we did not perform routine druj arthroscopy because of its limitation to visualize the foveal area which was usually filled with scar tissue, and a risk of cartilage damage when inserting the instrument to the narrow and tight joint space . Atzei et al.11 (2008) reported arthroscopic - assisted reattachment of the tfcc to the fovea using a suture anchor, resulting in significant improvement of grip strength and pain level . Iwasaki and minami17 (2009) introduced an arthroscopic outside - in suture technique without opening the capsule by creating a 2.9 mm osseous tunnel from the ulnar neck to the fovea under the fluoroscopic guide.18 they passed a suture into the tfcc through the osseous tunnel and tied it to the periosteum at the entrance of the tunnel . Although they reported favorable results, it seems inadequate to provide enough tension for maintaining the reattachment . Similarly, shinohara et al.19 (2013) and nakamura et al.20 (2011) described arthroscopically - assisted repair by means of a transosseous outside - in technique without exposure of the foveal site . Two separate small holes were made using 1.2 mm k - wires, and the pull - out suture was tied with tension . Unlike our technique, no attempt was made to debride the fibrous tissue and curettage of cartilage at the foveal site . We approached the tfcc foveal site through a mini - open incision on the volar capsule as described by atzei et al.11 we denuded the scar tissue and exposed the raw bone on the foveal site to facilitate healing of the reattached tfcc . After we made a suture loop at the peripheral portion of the tfcc through the opening of the volar capsule, fixation to the foveal insertion was attempted using either a transosseous pull - out suture or a knotless suture technique . The transosseous pull - out method is very effective in completing the knot with sufficient tension . However, we needed to make bone tunnels from the foveal site to the ulnar neck, which is technically demanding . Recently, we used a knotless suture anchor, which allows secure fixation while applying the sufficient tension . It has advantages of no need to make bone tunnels, no knot formation in the joint space, and requirement of a small skin incision . When a suture anchor was used,11 the knot was located inside the joint causing irritation to the surrounding tissues during wrist motion . Transosseous sutures, which make the knot outside the cortex at the ulnar neck, can also irritate the extensor carpi ulnaris and surrounding soft tissues.19 by using the knotless suture anchor, we did not observe surgical site pain caused by suture knot irritation . Nakamura et al.20 reported that the prognosis of the arthroscopic tfcc repair was not good if more than 7 months had elapsed after the trauma, probably due to a poor healing potential of the detached tissue . They performed all arthroscopic tfcc repairs without debriding the scar tissue, which may inhibit healing of reattached tfcc, particularly in patients with chronic tears . In contrast to the arthroscopic repair, they reported excellent results with an open repair technique even in chronic cases, suggesting that open debridement provides superior healing capabilities . Shinohara et al.19 also performed all arthroscopic repairs without debridement of the foveal site; however, they reported superior clinical results regardless of the elapsed time between the trauma and the surgery . They emphasized the choice of appropriate indication, which was traumatic tfcc foveal tears inducing moderate druj instability without an ulnar abutment . We also selected the patients for arthroscopic repair who had grade 2 druj instability without evidence of ulnar abutment . Our study showed a statistically significant improvement of pain and functional scores although the recovery of grip strength was not marked . The mean period of time from the injury to the operation was 12.2 months (range 436 months), and there was no significant difference of the postoperative prwe pain and dash score (p = 0.715 and p = 0.144, respectively) whether the time of surgery was more or <8 months from the trauma . We suggest that the selection of the patients with grade 2 dynamic druj instability is critical for favorable results after arthroscopic repair of tfcc foveal avulsion regardless of the time elapsed from injury . In delayed case, ligament atrophy with joint degeneration could be progressed, and healing potency of tfcc would be decreased conspicuously . Moreover, deterioration of druj instability with or without caput ulnar syndrome makes it difficult to obtain the satisfactory clinical result after arthroscopic repair only . So, druj reconstruction would be indicated if severely persistent instability was seen in physical examination and ulnar shortening osteotomy could be an another option by the effect of increasing the tension of distal radioulnar ligament.21 the limitations of the study were the small number of patients, its retrospective design, and its relatively short followup period . It is conceivable that the number of patients was too small to obtain a meaningful increase in grip strength after the operation . In addition, the short followup period may have affected the final grip strength because it might have been an insufficient amount of time for a notable recovery of muscle power . Arthroscopic repair of the tfcc foveal tear is a good modality in the management of druj dynamic instability providing satisfactory pain relief and functional improvement.
The dermal vascular changes have been evaluated by electron microscopy and shown to be due to change from arterial - type vessels to venules . The vascular changes may also precede the epidermal alterations, as shown in some studies . Morphometric evaluation of dermal vasculature in psoriasis has shown increased endothelial and luminal volume of vessels compared to control subjects . Studies utilizing native capillaroscopy have demonstrated reduction in microvessel density and length density after pulsed dye laser treatment . However, none of the earlier studies have compared vascular morphology between psoriasis and psoriasiform dermatitis . This study was aimed at evaluating the morphometric parameters of papillary dermal vessels in psoriasis and to compare these with psoriasiform lesions . In this study, skin biopsies from 25 cases of psoriasis and 25 of psoriasiform lesions were included . The diagnosis of psoriasis was confirmed on the basis of clinical features (pink to red papules with fine silvery scales and positive auspitz sign) in conjunction with characteristic histologic changes . The latter included: regular acanthosis with club - shaped rete ridges, suprapapillary epidermal thinning, hypogranulosis, confluent parakeratosis, spongiform pustules of kogoj and/or munro microabscesses . Psoriasiform lesions included in the study were: endogenous eczema: nine cases, hyperkeratotic eczema: seven cases, seborrheic dermatitis: three cases, nummular eczema: 2 cases, and one case each of allergic contact dermatitis, irritant contact dermatitis, lichen simplex chronicus, and pityriasis rosea . Serial sections were stained for cd34 (biogenex, usa) using the standard streptavidin - biotin - peroxidase method . Using image analysis software, the number, area, and length density of microvessel in dermal papillae were calculated from the cd34-stained sections . The microvessel density was calculated as number of vessels per unit (mm) papillary dermal area . Length density of microvessel (mm / mm or mm) was calculated by superimposition of cycloid grid on vertical sections of skin biopsy [figure 1]. Microvessel length density is defined as length of microvessels per unit dermal area and was calculated as 2i / l, where i refer to the number of intersections and l is total length of test lines . Ratio of microvessel area to the papillary dermal area was also calculated in both the groups included . Photomicrograph showing immunostaining for cd34 in a case of psoriasis with superimposed cycloid grid for morphometry (lsab, 200) appropriate statistical analysis was performed on the morphometric parameters between the two groups . On light microscopic evaluation of cd34-stained sections, biopsies from psoriasis showed much greater microvascular staining in the papillary dermis compared to psoriasiform dermatitis [figure 2]. Photomicrograph demonstrating the dermal vessels in psoriasis (a, lsab, 200) and psoriasiform dermatitis (b, lsab 200) morphometric analysis showed that length density of microvessels was significantly higher in psoriasis (3.731.45 mm) compared to psoriasiform lesions (2.991.99 mm, p value 0.03). Microvessel density, i.e., number of vessels per unit dermal area, was also higher in psoriasis (71.2840.05) than psoriasiform lesions (62.732.03), though the difference was not statistically significant . In contrast to these parameters, the ratio of microvessel area to papillary dermal area was similar in psoriasis (0.130.07) and psoriasiform lesions (0.140.11) [table 1]. Comparative analysis of dermal microvasculature in psoriasis and psoriasiform dermatitis these three morphometric vascular parameters are depicted in scatter plots [figures 3a c]. (a) scatter plots of vessel area per unit papillary dermal area; (b) microvessel density; and (c) length density in psoriasis and psoriasiform dermatitis psoriasis is an autoimmune disease affecting people of all ages, though there is a tendency for onset in early adulthood in patients with genetic transmission . Apart from epidermal changes, skin biopsies from patients with psoriasis demonstrate alterations in papillary dermis such as edema and dilated tortuous capillaries . These dilated capillaries are the source of bleeding points generated by gentle scraping of skin (auspitz sign). Few ultrastructural studies have shown that dermal capillaries in psoriasis have a wider lumen, bridged fenestrations with edematous areas in endothelial cytoplasm . These changes also correlate with enhanced cutaneous blood flow, including in the perilesional clinically unaffected skin . It has been proposed that vascular changes in the papillary dermis, including enhanced expression of adhesion molecules, permit leukocyte adhesion and assist in the establishment of inflammatory response . Barton et al ., in their study, demonstrated higher endothelial volume and luminal volume in the lesional psoriatic skin compared to uninvolved skin of patients with psoriasis as well as control subjects . The authors suggested that increase in these parameters was partly due to the increase in number of vascular profiles . In another study, computerized image analysis was performed to demonstrate the great degree of expansion of the vascular plexus in papillary dermis . The same study also showed significant endothelial proliferation in the papillary dermis of active psoriatic plaques . This study aimed at evaluating the vascular changes in psoriasis vis - - vis psoriasiform dermatitis . This comparison has not been performed in the available literature till date, to the best of our knowledge . We found a significantly higher length density of vessels in psoriasis compared to psoriasiform lesions (p<0.05). Microvessel density was also higher in psoriasis, though the difference did not reach statistical significance . These results indicate that though there is some vascular proliferation in response to inflammation in psoriasiform dermatitis, the tortuosity, and elongation of vessels reflected by length density is significant in psoriatic lesions only . With the advancing automation in diagnosis, these morphometric techniques might assist in differentiation between psoriasis and psoriasiform lesions, especially in early lesions of psoriasis . The vascular proliferation in psoriatic skin systemic therapy for psoriasis, such as methotrexate, cyclosporine, and tnf antagonists exert their therapeutic effect by immune modulation as well as interference with proangiogenic mediators . In addition, several molecules such as neovastat (inhibitor of endothelial cell proliferation) and inhibitors of vegf receptors are undergoing clinical trials . Hern et al ., performed native capillaroscopy and showed a significant reduction in the microvessel density, image area fraction, length, density and vessel image width after pulsed dye laser treatment of plaque psoriasis . Hence, the superficial microvascular bed in psoriatic skin is a legitimate and easily accessible target for antiangiogenic therapy . The present study demonstrates significant difference in the papillary dermal vasculature between psoriatic skin and psoriasiform dermatitis . These results, which need to be confirmed in further larger studies,
Rape is defined by the world health organization (who) as any sexual act or attempt to obtain sexual act, with unwanted sexual comments or advances against a person s sexuality using coercion (2). Brazilian law defines rape as embarrassing someone through violence or serious threat to have sexual intercourse or to perform or allow the practice of other libidinous acts (3). Between 1% and 12% of women, over 15 years of age have been sexually assaulted by unknown perpetrators (4). In the united states, the prevalence is 18% in adult women with an annual incidence from 0.3% to 1.1% (5), whereas in south africa, the prevalence is also high, but hardly ever measured (4). In brazil, the impact of sexual violence is multiplied when attacked women become pregnant and violence is passed from generation to generation (7). Less than 10% of cases of sexual assault are registered in police stations (8). The number of confirmed cases of rape using physical violence during practice increases when the perpetrators are unknown . The violence practiced by current partners is as prevalent as that perpetrated by strangers, but since cohesion is often not physical, it leaves no trace and therefore, the complaint is not performed (5). The assistance to victims of rape is divided into physical and psychological care (4, 810) and collection of evidence for criminal prosecutions (11). The collection involves physical, emotional and health examination and observations about body injuries including the genital area with collection of samples for dna analysis (4, 9). Given the above and the relevance of this topic, this study aimed to characterize rape committed against brazilian women and to identify possible associations between its occurrence and variables related to the victims, to perpetrators, to the aggression and to the resulting injuries . A retrospective study design was undertaken by the analysis of expert medical reports derived from medical forensic exams performed at the department of forensic medicine of the city of campina grande, pb, brazil, between january 2005 and december 2009 . The city of campina grande presented a considerable cultural, social and economic disparities, average monthly income of $110 per capita and human development index of 0.72 . About 886 forensic medical reports of sexual violence committed against female victims (children, adolescents, adults and elderly) were analyzed . The inclusion criterion was the crime of sexual abuse presented by the patient or her legal representative consistent with the provisions of articles 213 and 217-a of brazilian criminal law . Article 213 criminalizes as rape any non - consensual sexual act committed with the use of physical force or serious threat . Article 217-a, dealing with rape of vulnerable individuals, covers sexual acts against children under 14 years or against those of any age who cannot offer resistance or give consent (1). Information regarding socio - demographic variables (year, age, marital status and educational level) were collected related to the offenders (known or unknown; single or multiple and age) and aggression (location, time elapsed between the event and forensic examination, previous virginity, use of violence, body injuries and collection of material for dna testing). The information from the forensic medical reports were gathered and transferred to specific registration forms . Data analysis involved descriptive statistics (frequency distribution) and analytic statistics . To test the association between the occurrence of rape and other variables a process of bivariate analysis was conducted, using the exact versions of the nonparametric pearson s chi - squared test or fisher s exact test . The level of statistical significance was set at 5% with a confidence interval of 95% . This study followed ethical guidelines recommended by the brazilian legislation and was approved by the human research ethics committee of the state university of paraiba . A retrospective study design was undertaken by the analysis of expert medical reports derived from medical forensic exams performed at the department of forensic medicine of the city of campina grande, pb, brazil, between january 2005 and december 2009 . The city of campina grande presented a considerable cultural, social and economic disparities, average monthly income of $110 per capita and human development index of 0.72 . About 886 forensic medical reports of sexual violence committed against female victims (children, adolescents, adults and elderly) were analyzed . The inclusion criterion was the crime of sexual abuse presented by the patient or her legal representative consistent with the provisions of articles 213 and 217-a of brazilian criminal law . Article 213 criminalizes as rape any non - consensual sexual act committed with the use of physical force or serious threat . Article 217-a, dealing with rape of vulnerable individuals, covers sexual acts against children under 14 years or against those of any age who cannot offer resistance or give consent (1). Information regarding socio - demographic variables (year, age, marital status and educational level) were collected related to the offenders (known or unknown; single or multiple and age) and aggression (location, time elapsed between the event and forensic examination, previous virginity, use of violence, body injuries and collection of material for dna testing). The information from the forensic medical reports were gathered and transferred to specific registration forms . Data analysis involved descriptive statistics (frequency distribution) and analytic statistics . To test the association between the occurrence of rape and other variables a process of bivariate analysis was conducted, using the exact versions of the nonparametric pearson s chi - squared test or fisher s exact test . The level of statistical significance was set at 5% with a confidence interval of 95% . This study followed ethical guidelines recommended by the brazilian legislation and was approved by the human research ethics committee of the state university of paraiba . The incidence of rape was 32.8% (n = 291), with the largest percentage of cases (22.7%) being recorded in 2005 . The average age of victims was 15.68 years (6.36), with minimum of two years and maximum of 68 years . Most were between 10 and 19 years (89.9%), were single (98.8%) and had low educational level (86.9%). There was an association between rape and marital status (p = 0.02) (table 1). Distribution of victims of rape according to sociodemographic variables a small number of medical reports contained information regarding the age of the aggressor, but reveal that the majority had 20 years or more (69.2%) with mean of 27.4 years (11.0), minimum of 16 years and maximum 50 years . When evaluating the relationship between victim and sex offender, in 84.2% of cases the perpetrators were known to the victims, being the current partner responsible for 31.8% of cases . Sex offenders with blood ties (biological father, uncle and brother) totaled 5.7% of cases . Regarding the number of offenders, association between rape and variables related to aggressor among the cases where the number of attackers was multiple, 12 cases involved two perpetrators, one involved three perpetrators and one case involved five perpetrators . An association was observed between rape and the age of the sex offender (p = 0.03), type of relationship (p = 0.001, pr = 2.32 (1.62 to 3.33)) and known offenders (p = 0.001). Information concerning the site of rape occurrence was not present in 76.4% of medical reports . In situations in which the site of rape occurrence was identified, most rape occurrences were recorded in the home environment (n = 35; 49.3%), and eighteen cases (51.4%) were recorded in the victim s residence and seventeen (48.6) were recorded in the aggressor s residence . Regarding the date of occurrence, 70.5% of cases had 20 days or more from the completion of the forensic examination and 80.9% of victims were virgins before the rape . The presence of violence during the rape practice was found in 72.3% of cases, but physical injuries were identified in only 5.2% of victims . In only 34.8% of occurrences, collection of material for dna testing was performed (table 3). There was an association between rape and date of occurrence (p = 0.001), previous virginity (p = 0.001) and violence during practice (p = 0.001). The data presented here demonstrate that victims of violence do not have a homogeneous profile and differ regarding the sociodemographic characteristics and those related to the event . Of the 886 analyzed cases of sexual violence, the existence of rape was found in about one third of victims, a result higher than that reported among american women (12). The divergence between results is due to the collection site of studies, since while this research was conducted at the institute of forensic medicine and dentistry, institution for which brazilian victims of sexual violence are referred, another study was carried out in a hospital unit . About 6.8 million cases of rape and physical assault in the united states are estimated each year, with 2.6 million resulting in physical injuries (9). When the perpetrator is known to the victim, the complaint is inhibited, reflecting a false prevalence . Women are afraid of reprisals and feel ashamed, humiliated and guilty (13). The lack of information; however, prevents measuring the frequency of this fact, thereby contributing to under - reporting (14). Most of the victims were 19 or younger, a result similar to that observed in the united states, with predominance of adolescent victims aged from 12 to 17 yr (5, 9). Hospital revealed mean age of 26.2 years (9), which is higher than that found in this work . Brazilian researchers found that the prevalence of victims aged under 19 yr ranges from 47.6 (16) to 77% (15). The prevalence of this crime in this age group may be related to the greater consumption of alcohol and illicit drugs, increased frequency of relationships with others, greater exposure to domestic violence, greater public exposure and finally the earlier sexual development primarily driven by the media (17). Most victims had low educational level, a result similar to that seen in the united states (12) and brazil (16). In this research, predominance of young victims, mostly students was found, corroborating the findings of other brazilian authors (16). Aggressions against women are mainly committed by intimate or known people (8). In this study, 84.2% of offenders were known to the victims, with predominance of the current partner, corroborating previous findings (5,11,14,16). In the united states, more than half of women reported that at some point in their lives, they had been victims of physical aggression or sexual assault by their current partners (12). It could be inferred that while father, stepfather, uncles and brothers are the most frequent aggressors against children, spouse or intimate partners are the most frequent perpetrators against adult women . In 6.2% of cases, more than one perpetrator participated in the violence, which is in accordance with literature that reports a low frequency of multiple aggressors (11). In this study, the number of offenders varied from two to five . Psychological sequelae may be more severe when the crime involves multiple perpetrators (1). A significant number of cases of rape occurred at home, both the victim and the aggressor, diverging from data described in literature, where most cases of sexual violence occurs on public roads (16). Women attacked at the domestic environment are those that least denounced violence to authorities, since most are abused by known people (8). Many victims of this research had suffered aggression for more than 20 days from the date of the forensic medical examination . The literature reports that most women present complaint within 72 h after sexual violence (5). However, if the aggressor is known to the victim, it takes longer to seek emergency care . On the other hand, when the victim does not know the aggressor, the seek for health services is faster, as identified by brazilian researchers who reported that two thirds of women sought health services within 24 h after sexual violence and emergency care was performed within the first 72 h in 87.6% of women (16). More than two thirds of the victims reported the use of force - (fists or choking, knife, gun, restraints, blindfold, or other means), confirming previous findings (16). When violence involves children, usually there is use of physical force, but among adolescent and adult women, sexual violence requires physical violence because victims have greater physical size and strength to resist . Physical injuries confirm violence during practice, thus becoming an important material evidence for criminal cases, but their absence does not eliminate the possibility of crime (18). In this study, few victims exhibited physical injuries resulting from sexual abuse during forensic examination, which was also observed by other authors (11). However, this fact may be related to the time elapsed since the sexual violence, which in most cases, the victims were examined for sexual violence only after 20 days . Another possible explanation for the absence of injuries is the use of weapons for coercion, preventing occurrence of fights with physical signs of resistance (14). Research conducted in south africa showed that the confirmation of sexual violence is more common when there is presence of physical injuries and when dna collection is performed (4). In this study, the number of victims who had material collected for dna testing was extremely low . It was not possible to identify why this occurred; however, it may be due to the fact that there were few victims who immediately sought the institute of forensic medicine for collecting material . The first is the fact that it was developed in a unique institute of forensic medicine of a single city . Another limitation was the large number of medical reports with missing information (unfilled, unanswered or inconclusive); however, the findings described here faithfully depict the characteristics of perpetrators and women victims of sexual violence in campina grande, brazil; being extremely important for public policy planning and resource allocation in public safety . Over one third of women were victims of rape, predominantly adolescents, unmarried and with low educational level . The aggressors were known to the victims, and acted alone in most situations, making use of physical violence . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc .) Have been completely observed by the authors.
Malpositioning of an endotracheal tube (ett) can cause serious complications in airway management and mechanical ventilation . When the ett is inserted deeply, there is a risk of endobronchial intubation, resulting in atelectasis, hypoxemia, and barotrauma . In contrast, an insufficiently inserted ett causes accidental extubation . To confirm proper positioning of the ett after tracheal intubation, various methods have been introduced, including the use of a predetermined depth (21 cm for females and 23 cm for males) or guide marks on the ett at the level of the vocal cords, chest auscultation, and the cuff ballottement technique at the suprasternal notch . The cuff ballottement test, which confirms that the inflated cuff is positioned at the suprasternal notch with squeezing or inflating a pilot balloon, has been reported to be a simple and reliable method to confirm the correct depth of the ett . In addition, vocal cord - related complications are possible due to the ett cuff, and thus it should be placed 15 mm below the vocal cord to prevent recurrent laryngeal nerve injury . However, the length of recently developed ett cuffs is generally around 40 mm (table 1) and thus, in patients with a short distance between the vocal cord and the suprasternal notch, ett positioning using the cuff ballottement test can cause pressure on the vocal cord and recurrent laryngeal nerve, and the risk of intralaryngeal placement cannot be excluded in severe cases ., we hypothesized that ett placement using the cuff ballottement test might be related to vocal cord injury and retrospectively investigated the distance from the part 15 mm below the vocal cord to the suprasternal notch (vsd-15) using computed tomography (ct) imaging . The length between the thyroid notch and the suprasternal notch (thyroid notch - to - suprasternal notch distance [tsd]) was also examined to evaluate the correlation with vsd-15 with consideration given to patient height, weight, sex, and age . This retrospective study was approved by the institutional review board of seoul metropolitan government seoul national university boramae medical center (no ., the data of 778 adult patients who underwent neck ct from january 2010 to december 2012 were retrieved from the hospital database . Patients were excluded if they had diseases or masses that could affect the contour of the larynx, the upper trachea, or the suprasternal notch, based on the examination of ct images by radiologists . Patients who had ambiguous anatomical outlines due to poor - quality ct images were also excluded . Scout images of the frontal and lateral neck obtained before axial ct images of the neck were acquired . The vocal folds are the narrowest part of the larynx and the infraglottic space widens and connects with the cavity of the trachea . Vsd-15 was estimated as the distance from a point 15 mm below the vocal cord to the suprasternal notch, superior border of the manubrium, between the clavicular notches at the frontal neck scout image (fig . The tsd was assessed as tsd, the superior border of the manubrium, at the lateral neck scout image (fig . Vsd-15 means the safe proximal margin for endotracheal tube cuff placement above the suprasternal notch . Tsd is chosen as a predictive factor because the vocal cord exits on the posterior surface of the thyroid cartilage in adults and thyroid cartilage is easily detected externally . Vsd-15 = the distance from 15 mm below the vocal cord to the suprasternal notch, tsd = the distance from the thyroid notch to the suprasternal notch . Statistical analyses were performed using the ibm statistical package for social sciences statistics software program (ver . Data are expressed as means (standard deviation). To assess the reliability of vsd-15 and tsd measurements, the intraclass coefficient (icc) was calculated . To investigate the relationship between vsd-15 and other variables, such as age, sex, height, and tsd, linear regression was used . Prior to analysis, any sex difference in vsd-15 was analyzed using student t test . Pearson correlation coefficient was used to analyze continuous variables and multiple linear regression was used to analyze the effect of each variable on vsd-15 . In the multiple linear regression model, we included variables with a p value <0.05 in a single variable analysis . To avoid the problem of multicollinearity, we included independent variables in the models . A p value <0.05 was considered to indicate statistical significance . In total, 778 patients underwent neck cts from january 2010 to december 2012 . Of them, 255 patients who had diseases or masses that could affect the contour of the larynx, the upper trachea and the suprasternal notch were excluded, and 96 were excluded because of ambiguous outlines on ct images . The iccs, a measure of reliability, were 0.921 for vsd-15 and 0.930 for tsd . Flow diagram of patient selection for image analysis . Of 778 patients who underwent neck ct, 255 patients were excluded because of diseases or masses that could affect the contour of the larynx, the upper trachea, and/or the suprasternal notch, and 96 patients were also excluded because of the ambiguous outlines in the ct images . The analyzed patients, 47 patients (11.0%) showed a vsd-15 shorter than 45 mm . Significantly correlated with tsd (r = 0.778, p <0.001) and height (r = 0.312, p <0.001), and it was inversely correlated with age (r = 0.321, p <0.001). However vsd-15 = the distance from 15 mm below the vocal cord to the suprasternal notch, tsd = the distance from the thyroid notch to the suprasternal notch . In the multiple linear regression models, tsd, height, and age showed statistically significant correlations with vsd-15 (table 3). From the results, a prediction model for vsd-15 was as follows: associations of vsd-15 with independent variables by multiple linear regression analyses . Our data suggest that patients with relatively short vsd-15 values may be susceptible to vocal cord injury following ett placement using the cuff ballottement test . Vsd-15 can be predicted from tsd, height, and age . In general, when the ett tip is positioned at the level of the mid - trachea, the risk of endobronchial intubation or inadvertent extubation decreases . The trachea in adults extends from the cricoid cartilage at the level of c6 to the carina at the level of t4 . The suprasternal notch lies at the vertebral level of t12, slightly above the middle trachea, and thus if the tt cuff is detected just above the suprasternal notch during the cuff ballottement test, the tip of the ett would be positioned at the level of the mid - trachea . According to a previous study, the cuff ballottement test is a reliable method that predicts when the tip of the ett lies in the desired position, 3 to 7 cm from the carina, at the level of t34 . Proper positioning of the ett has mainly focused on the avoidance of endobronchial intubation or inadvertent extubation, but vocal cord - related complications following tracheal intubation can also be problematic . Hoarseness and sore throat are common postoperative complaints, with incidences between 15% and 49% . Furthermore, vocal cord paralysis is one of the most serious complications following tracheal intubation, resulting from mechanical damage or nerve injury . A pressurized tt cuff can damage the vocal cord, and this is related to ett size, cuff pressure, and direct recurrent laryngeal nerve injury . In particular, the anterior ramus of the recurrent laryngeal nerve is placed about 6 to 10 mm below the vocal cords, and is vulnerable to compression between the thyroid cartilage and the ett cuff . Furthermore, the vocal cord is the narrowest portion of the larynx and the infraglottic cavity expands anterolaterally, connecting to the trachea . If the ett cuff is placed immediately below the vocal cord, the cuff exerts a pressure on the recurrent laryngeal nerve, resulting in sore throat, hoarseness, or vocal cord paralysis . Thus, it has been advocated that the upper end of the ett cuff should be placed 15 mm below the vocal cord . When the cuff ballottement test is used, the ett cuff is placed around the suprasternal notch . In some cases, the major part of the cuff could be placed above the suprasternal notch and immediately below the vocal cord, and patients with a short vsd-15 are susceptible to vocal cord injury . Additionally, newly developed ett cuffs tend to have a cylindrical shape to provide a large contact area for improving tracheal sealing, but it can induce more pressure on the vocal cord . To predict vsd-15, the safe proximal margin for ett cuff placement, we evaluated the correlation between vsd-15 and possible variables, including height, weight, age, and tsd . Tsd was chosen as a predictive factor because the vocal cord exits on the posterior surface of the thyroid cartilage; the lower end of the epiglottis is attached to the middle of the posterior surface of the thyroid cartilage in adults; and the thyroid cartilage is readily detected externally . In the present study although the underlying mechanism is unclear, it is assumed that laryngeal tissues might degenerate and shrink with age . According to a previous study, the risk of vocal cord paralysis was increased threefold in patients age 50 or older . One of their explanations is that the age - degenerated laryngeal system may be more vulnerable to acute inflammation and circulatory insufficiency due to the tt cuff pressure . Additionally, vsd-15 was correlated with height in agreement with previous reports which showed a correlation between airway length and height . Thus, height was included in the multiple linear regression models . In the present study, some patients had vsd-15 values shorter than 45 mm . Based on our findings, in patients with a relatively short vsd-15, the cuff ballottement test may cause the cuff to be placed in the vicinity of the vocal cord, which might cause vocal cord complications . Furthermore, it was found that these patients have a higher risk of endobronchial intubation because the thyrosternal length, which is tsd in our study, has been suggested to predict tracheal length . Thus, we should keep in mind that patients who are predicted to have a short vsd-15 are likely to suffer from vocal cord injury and endobronchial intubation, and a smaller tt with a shorter cuff length could be considered within the clinically acceptable range in these patients . First, we did not assess the relationship between vsd-15 and the incidence of postoperative sore throat, hoarseness, or vocal cord paralysis . Further studies are needed to evaluate the correlation between the vocal cord - to - suprasternal notch distance and postoperative laryngeal complications . Second, our findings are restricted to asians, although airway length does not differ between western and oriental individuals according to previous studies . In conclusion, the cuff ballottement test should be used cautiously in patients who are predicted to have a relatively short vsd-15 . Future clinical evaluations of the relationship between the vocal cord - to - suprasternal notch distance and postoperative vocal cord complications are required . The authors would like to thank sohee oh, phd (medical statistician, department of biostatistics in smg - snu boramae medical center), for statistical advice and the electronic medical record team of our hospital for supporting the data review.
Struma ovarii is an ovarian teratoma, represented in more than 50% by thyroid tissue . Five percent of struma ovarii cases have been proven to be malignant and, as in the thyroid gland, papillary thyroid carcinoma is the most common histotype arising in struma ovarii . Because of the unusual occurrence of this tumor, its management and follow - up after pelvic surgery is still controversial . Usually, total thyroidectomy followed by radioiodine treatment is the choice treatment in metastatic malignant struma ovarii, while these procedures are still controversial in non - metastatic thyroid cancer arising in struma ovarii . We report a female with follicular variant of papillary thyroid carcinoma arising in struma ovarii . After pelvic surgery, thyroid morphofunctional examinations were performed and a single nodular lesion in the left lobe was discovered . Radioiodine - ablation of residual thyroid tissue was performed and levothyroxine mildly - suppressive treatment was started . A more aggressive treatment should not be denied for malignant struma ovarii without any evidence, even when apparently confined into the ovary . However, in selected cases, aggressive treatment may be advisable to decrease the risk of recurrence and to allow an accurate follow - up . Germ cell tumors represent 15 - 20% of ovarian cancers and most of them are mature cystic teratomas (1). Struma ovarii is a highly specialized ovarian teratoma, chiefly represented by thyroid tissue (2). In fact, while 5 - 15% of teratomas contains small foci of thyroid tissue, this latter should represent more than 50% in a struma ovarii (1). Approximately, 5% of struma ovarii has been proven to be malignant and metastases are uncommon (2). Fairly good prognosis characterizes these patients: survival rate of patients in follow - up at 5, 10 and 25 years is 92%, 85% and 79%, respectively (3). Long - term follow - up is recommended in all cases (4). As in the thyroid, papillary carcinoma is the most common malignant histotype (70%, 1/3 represented by its follicular variant) arising in struma ovarii; while follicular carcinoma is observed in the remaining 30% (3 - 5). Malignant struma ovarii generally occurs in patients in the fifth and sixth decades of life (5), it is unilateral (94%) and more frequently involves the left ovary (3, 5). Functional thyroid alterations are unusual and overt hyperthyroidism is reported in 5 - 8% of cases (5). The most common clinical presentation of struma ovarii is a pelvic mass (3); lower abdominal pain, abnormal vaginal bleeding or menstrual irregularities and ascites may be associated (5). However, the tumor is often unexpectedly diagnosed during abdominal / pelvic ultrasound or surgery (3, 5). Abdominal hysterectomy and bilateral salpingo - oophorectomy with omentectomy unilateral salpingo - oophorectomy is the preferred treatment for women who require preservation of fertility . Being this tumor fairly uncommon, there is a lack of diagnostic and treatment guidelines (4, 6 - 8). After initial surgery, some authors suggest that the management of malignant struma ovarii may be similar to that of other germ cell tumors (1); other authors suggest, as in differentiated cancer of thyroid gland management, to perform a thyroidectomy followed by radiotherapy with 131 isotope of iodine (rai) (9) and levothyroxine suppressive therapy (3, 4, 7, 8), although restricted to patients with recurrence or residual disease (2, 4, 5). No consensus has been reached in performing prophylactic total thyroidectomy and the treatment strategy for nonmetastatic malignant disease remains controversial . We present a case of 30-year - old female with follicular variant of papillary thyroid carcinoma arising in struma ovarii and concomitant differentiated cancer of thyroid gland . A 30-year - old woman was admitted to the emergency room because of persistent abdominal pain in the right side, appeared some day before, during a shift work as agricultural laborer . An accurate medical history ascertained that she had an increased steady weight (109 kg; bmi = 40 kg / m), but she was in apparently good health also showing customary appetite and regular menses, before the appearance of pain . Physical examination revealed the presence of a large mass, palpable in hypogastric region and in right lower abdominal quadrant . To better define the diagnosis, the physician required blood sampling for routine tests, gynecological counseling and an abdominal computer tomography (ct). The abdominal ct confirmed the presence of a mass measuring 10 x 6 cm, in the right ovary . In particular, an ovariian cyst (7, 3 x 3 cm) along with a portion of the fallopian tubes of 4 cm and the vermiform appendix were removed . Peritoneal washing and multiple peritoneal biopsies in nearby areas (douglas, bladder, bilateral paracolic gutter, and diaphragm) were also performed . Histological examination revealed the presence of a follicular variant of papillary thyroid carcinoma (0.9 cm) harbored in a mature teratoma . The cancer was localized and the right ovary capsule was not infiltrated by the tumor . Based on union for international cancer control (uicc) 2002, appendix showed the features of chronic follicular inflammation and the remaining tissues were disease - free . After surgery, functional examination of thyroid gland revealed serum iodothyronines levels in the normal range, thyroid ultrasonography showed a normal glandular volume with patchy echo - structure and, in the left thyroid lobe, the presence of a 23, 4 x 14 x 9 mm hypoechogenic nodular lesion . An ultrasound assisted fine needle aspiration of this thyroid lesion was performed and cytological examination showed the presence of dense colloid in particles and small groups of thyrocytes with notes of oxyphilic metaplasia and anisonucleosis; this sample was classified as follicular lesion with oxyphilic cells of undetermined significance (category iii according to the bethesda system, 2009) (10). So far, in presence of follicular lesion and for an appropriate follow - up of papillary carcinoma arising in struma ovarii, the patient underwent total thyroidectomy (11, 12). Histological examination revealed that the nodule examined in the left lobe was benign, but showed a papillary micro - carcinoma in the right lobe (diameter = 0.2 cm; pt1a) within a hyperplastic thyroid characterized by abundant oxyphil cells . The ablation of residual thyroid tissue was performed by a dose of 30 mci of 131i according to meas et al . Whole body scan after therapeutic dose showed two focal areas of strong uptake in the anterior region of the neck, in right superior paramedian and left inferior paramedian areas, due to minimal glandular remnants; no area of uptake in the pelvis or elsewhere was notice . Mu / l and serum thyroglobulin (tg) was 1.24 mg / dl, in the absence of anti - thyroglobulin antibodies (anti - tgab). Levothyroxine treatment was started (1.6 g / kg / day) and thyroid hormones and tsh were evaluated after four months . Six months after total thyroidectomy, patient underwent neck ultrasonography and tg evaluation, which showed no evidence of recurrences . Patient is up to now in follow - up for both struma ovarii and differentiated thyroid cancer according to european thyroid association guidelines (12). First described at the end of 21th century, struma ovarii is a rare tumor representing less than 1% of all ovarian tumors (6, 14) and 2.7% of all dermoid tumors (6). A diagnostic and therapeutic characterization of struma ovarii is needed as it may harbor a differentiated thyroid cancer (1). Malignant struma ovarii has been, in fact, replaced with the more appropriate (tcaso), namely when histological features support the existence of a well - differentiated thyroid carcinoma (3, 14, 15). Molecular evidence that malignant struma ovarii and differentiated thyroid cancer may share similar pathogenic events (3, 16) has been presented . Approximately 70% of follicular cell - derived thyroid carcinomas are associated with activating mutations of braf, ras, ret and ntrk1 (3, 4, 16). Braf mutations have been described in 29 - 69% of primitive papillary thyroid cancer and in particular v600e missense mutation type is the most common alteration in sporadic papillary carcinoma (3, 17). Also in four out of six malignant struma ovarii and in none of nine benign struma ovarii, braf mutations were observed in the schmidt s study (3, 4). The concurrent presence of the same genetic alteration (i.e. Braf mutations), in thyroid neoplasia and in struma ovarii would be intriguing in that it may support the hypothesis of a multifocal thyroid neoplasia in two distant sites (13, 16). Autonomous histopathological criteria have not been established for thyroid carcinoma arising in struma ovarii and, so far, the diagnosis of malignant struma ovarii follows the guidelines for the diagnosis of primary thyroid carcinoma (3). Evidence also indicates that patients with malignant struma ovarii should be treated as those having differentiated cancer in the thyroid gland (3, 10). In patients with thyroid cancer confined to the ovary, pelvic surgery has been considered sufficient and prophylactic thyroidectomy not recommended (1, 2, 14); in these patients follow - up ensues as in non - aggressive dtc (1, 14). Fine needle aspiration cytology has been considered appropriate when thyroid nodular lesions would have been detected at ultrasound (1, 4, 14). Noticeably, however, a thyroid carcinoma concurrent with malignant struma ovarii is increasingly detected (14, 18) and the treatment of this association is, as yet, not properly defined . In fact, a more extensive treatment, e.g. Total thyroidectomy and rai, after pelvic surgery, is only recommended for malignant struma ovarii with extra - ovarian spread or distant metastasis (1 - 3). In patients with extra - ovarian or metastatic disease, evidence suggests that radioiodine ablation and thyrotropin suppression are associated with increased disease - free survival (3, 7). According to another study, patients who underwent total thyroidectomy and radio ablation after pelvic surgery were free of recurrence even after 36 years (7, 8). On the contrary, a recurrence rate of 50% was associated with the conservative treatment of patients (8). Moreover, a delayed appearance of distant metastasis has been described three years after the treatment in patients with struma ovarii who underwent pelvic surgery alone (18). A not negligible fraction (6%) of patients with longer follow up, developed distant metastases after one, three and seven years (15). Two - thirds of metastasis occurred at the time of disease recurrence, rather than at first presentation (7); in fact, a number of cases were diagnosed as malignant only following the detection of metastatic recurrences (7). In this clinical case high prevalence of occult thyroid papillary microcarcinoma has been described in autoptic studies (36%) and/or following surgery for benign conditions (2 - 24%) (19). However, some of these microcarcinomas may represent more aggressive varieties of disseminated tumor cells (dtcs) (17). Indeed, papillary microcarcinoma, although infrequently, may show significant differences in clinical behavior, sometimes being very aggressive, despite small primary tumor size (17). (13), the contemporary presence of two possibly related thyroid cell - derived tumors, although with low risk stratification, may be considered as a multifocal expression of the same pathogenic alteration . They suggested that a threshold of more than one centimeter as sum of all foci may require a more cautious seeking of thyroid tissue lesions (13). In this view, american thyroid association guidelines (20) concerning dtc follow - up should be reinterpreted as being the struma ovarii and papillary carcinoma (any size) a multifocal expression of the same tumor . In conclusion, a treatment including thyroidectomy followed by radioablation should not be denied a priori, but in selected cases may be advisable to decrease the risk of recurrence and to allow an accurate follow - up (7).
We enrolled 1,047 consecutive caucasian patients referred to coronary angiography for the evaluation of stable cad solely on a clinical indication . Coronary angiography was performed as described previously (4); coronary stenoses 50% were considered significant (5,6). The mets was diagnosed according to american heart association revised national cholesterol education program adult treatment panel iii criteria (7). The ethics committee of the university of innsbruck approved the study; all participants gave written informed consent . Analytical procedures were performed on a cobas integra 800 (roche, basel, switzerland), as described previously (4,8). Sample - size calculations showed that assuming an sd of 1.5 times the population mean, 393 patients would be needed per study group to detect a between - group difference of crp of 20% with a power of 80% at an fault of 0.05 . Analytical procedures were performed on a cobas integra 800 (roche, basel, switzerland), as described previously (4,8). Sample - size calculations showed that assuming an sd of 1.5 times the population mean, 393 patients would be needed per study group to detect a between - group difference of crp of 20% with a power of 80% at an fault of 0.05 . Significant cad at angiography was present in 564 patients (55.8%); its prevalence was higher in patients with mets than in subjects without mets (59.5 vs. 52.8%; p = 0.034); adjustment for age, sex, ldl cholesterol, smoking, cardiovascular medications (statins, aspirin, ace inhibitors / angiotensin receptor blocking agents and -blocking agents), and crp confirmed this result, with an odds ratio (or) of 1.49 (95% ci 1.121.98; p = 0.007) for mets patients . The low hdl cholesterol (or 1.57 [95% ci 1.112.22]; p = 0.011) and the high - glucose traits (1.33 [1.021.73]; p = 0.038) proved significantly and independently of the above covariates associated with significant cad, whereas high triglycerides (p = 0.082), large waist (p = 0.826), and high blood pressure criteria (p = 0.145) were not independently associated with significant cad . Crp was significantly higher in patients with mets than in subjects without mets (0.46 0.62 vs. 0.35 0.49 mg / dl; p <0.001). In contrast, crp did not differ significantly between patients with significant cad and subjects without significant cad (0.40 0.59 vs. 0.39 0.52 mg / dl; p = 0.706). Crp also was similar in subjects with any atherosclerotic lesion at angiography compared with subjects with completely normal coronary arteries (0.41 0.57 vs. 0.36 0.50 mg / dl; p = 0.325). Furthermore, crp was not associated with significant cad in a multivariate model adjusting for age, sex, ldl cholesterol, smoking, cardiovascular medications, and presence of mets (standardized adjusted or 0.97 [95% ci 0.761.25]; p = 0.822). Considering both mets and significant cad, crp was significantly higher in patients with mets, both among those without significant cad (0.45 0.50 vs. 0.36 0.53 mg / dl; p <0.001) and among those with significant cad (0.47 0.69 vs. 0.34 0.45; p = 0.001). In contrast, crp did not differ between patients with significant cad and those without significant cad among subjects without mets (p = 0.869) or among subjects with mets (p = 0.411). Ancova, adjusting for age, sex, ldl cholesterol, smoking, and cardiovascular medications, confirmed that mets (f = 11.74; p = 0.001) but not significant cad (f = 0.01; p = 0.983) was significantly associated with crp . Univariately, serum crp was significantly higher in patients who fulfilled the large waist (p <0.001), the low hdl cholesterol (p <0.001), the high blood pressure (p = 0.016), and the high glucose (p <0.001) criteria but not in patients who fulfilled the high triglyceride criterion (p = 0.352) compared with patients who did not fulfill the respective mets criteria . When all mets traits were entered simultaneously into one ancova model, only low hdl cholesterol proved associated with crp (f = 44.19; p <0.001) independently of age, sex, ldl cholesterol, smoking, major cardiovascular medications, and of all other mets criteria . Crp increased significantly (ptrend <0.001) with an increasing number of mets traits (fig . 1a) after adjustment for age, sex, smoking, ldl cholesterol, and major cardiovascular medications . Further adjustment for the high waist (f = 11.66; p = 0.001), the high glucose (f = 14.18; p <0.001), the high blood pressure (f = 17.94; p <0.001), and the high triglyceride (f = 32.81; p <0.001) traits rendered this relationship virtually unchanged . In contrast, the positive association between the number of metabolic traits and crp was no longer significant (fig . 1b) after adjustment for the low hdl cholesterol criterion (f = 0.87; p = 0.352). A: relationship of the number of mets components and crp adjusted for age, sex, ldl cholesterol, smoking, and major cardiovascular medications . P value is given for the association of crp with the number of mets components . Significant cad at angiography was present in 564 patients (55.8%); its prevalence was higher in patients with mets than in subjects without mets (59.5 vs. 52.8%; p = 0.034); adjustment for age, sex, ldl cholesterol, smoking, cardiovascular medications (statins, aspirin, ace inhibitors / angiotensin receptor blocking agents and -blocking agents), and crp confirmed this result, with an odds ratio (or) of 1.49 (95% ci 1.121.98; p = 0.007) for mets patients . The low hdl cholesterol (or 1.57 [95% ci 1.112.22]; p = 0.011) and the high - glucose traits (1.33 [1.021.73]; p = 0.038) proved significantly and independently of the above covariates associated with significant cad, whereas high triglycerides (p = 0.082), large waist (p = 0.826), and high blood pressure criteria (p = 0.145) were not independently associated with significant cad . Crp was significantly higher in patients with mets than in subjects without mets (0.46 0.62 vs. 0.35 0.49 mg / dl; p <0.001). In contrast, crp did not differ significantly between patients with significant cad and subjects without significant cad (0.40 0.59 vs. 0.39 0.52 mg / dl; p = 0.706). Crp also was similar in subjects with any atherosclerotic lesion at angiography compared with subjects with completely normal coronary arteries (0.41 0.57 vs. 0.36 0.50 mg / dl; p = 0.325). Furthermore, crp was not associated with significant cad in a multivariate model adjusting for age, sex, ldl cholesterol, smoking, cardiovascular medications, and presence of mets (standardized adjusted or 0.97 [95% ci 0.761.25]; p = 0.822). Considering both mets and significant cad, crp was significantly higher in patients with mets, both among those without significant cad (0.45 0.50 vs. 0.36 0.53 mg / dl; p <0.001) and among those with significant cad (0.47 0.69 vs. 0.34 0.45; p = 0.001). In contrast, crp did not differ between patients with significant cad and those without significant cad among subjects without mets (p = 0.869) or among subjects with mets (p = 0.411). Ancova, adjusting for age, sex, ldl cholesterol, smoking, and cardiovascular medications, confirmed that mets (f = 11.74; p = 0.001) but not significant cad (f = 0.01; p = 0.983) was significantly associated with crp . Univariately, serum crp was significantly higher in patients who fulfilled the large waist (p <0.001), the low hdl cholesterol (p <0.001), the high blood pressure (p = 0.016), and the high glucose (p <0.001) criteria but not in patients who fulfilled the high triglyceride criterion (p = 0.352) compared with patients who did not fulfill the respective mets criteria . When all mets traits were entered simultaneously into one ancova model, only low hdl cholesterol proved associated with crp (f = 44.19; p <0.001) independently of age, sex, ldl cholesterol, smoking, major cardiovascular medications, and of all other mets criteria . Crp increased significantly (ptrend <0.001) with an increasing number of mets traits (fig . 1a) after adjustment for age, sex, smoking, ldl cholesterol, and major cardiovascular medications . Further adjustment for the high waist (f = 11.66; p = 0.001), the high glucose (f = 14.18; p <0.001), the high blood pressure (f = 17.94; p <0.001), and the high triglyceride (f = 32.81; p <0.001) traits rendered this relationship virtually unchanged . In contrast, the positive association between the number of metabolic traits and crp was no longer significant (fig . 1b) after adjustment for the low hdl cholesterol criterion (f = 0.87; p = 0.352). A: relationship of the number of mets components and crp adjusted for age, sex, ldl cholesterol, smoking, and major cardiovascular medications . P value is given for the association of crp with the number of mets components . From our data, we conclude that among angiographied coronary patients crp is strongly associated with the mets but not with angiographically characterized coronary atherosclerosis . Specifically, the overall association of the mets with crp is driven by the low hdl cholesterol feature . Data from the literature on the association of crp with cross - sectionally determined cad are controversial . This observation likely reflects the fact that inflammation is not associated with plaque burden itself but rather with plaque vulnerability and rupture . Thus, our data do not contradict the numerous reports on an association between crp and clinical atherothrombotic events . Further, our data show that the low hdl cholesterol mets feature drives the overall association between the mets and crp; crp was no longer associated with the number of mets traits when adjusted for hdl cholesterol . These data fit into the notion that hdl particles, besides their crucial role in reverse cholesterol transport, also protect the artery wall through anti - inflammatory mechanisms (13). Thus, crp is strongly associated with the mets but not with angiographically diagnosed coronary atherosclerosis . The overall association of the mets with crp is predominantly driven by the low hdl cholesterol feature, a paramount predictor of vascular events.
When i hear the word quantitation, what comes to mind is the contrast between two famous 19th - century german scientists, alexander von humboldt and carl gauss, as described in measuring the world: a novel (kehlmann, 2009). Von humboldt was a great prussian explorer who made many discoveries, including electric eels, in his expeditions to south and central america, which included the casiquiare canal, which connects the orinoco and amazon rivers . A man of extraordinary energy, he was the consummate quantitater he measured the heights of mountains and the numbers of lice on the heads of natives . Gauss, as surveyor and astronomer for the kingdom of hannover, was also a quantifier, but with a different style . Charged with devising a practical method to survey on a spherical earth, he laid the foundation for a school of geometry that culminated in the work of riemann and the notion of curved space - time in einstein's general theory of relativity . The lesson here is that when you have a theory, measurement becomes very important and intellectually challenging . Have all the relevant parameters been measured? Have all the confounding complications been considered? Does the theory really fit to the data? How accurately have the underlying quantities (parameters) been measured? What is a model? What is the difference? In biology, we usually distinguish three types of model: conceptual models, mathematical models, and simulations (bowne - anderson et al ., 2013). They all serve the same general purpose: to explain more complex phenomena in terms of more elementary processes or molecular interactions . This can be in the form of a cartoon or picture that conveys the author's idea or explanation of his or her results . But can it really explain the data? Usually, more work is needed to establish the plausibility of a conceptual model . A mathematical model is the development of the conceptual model into a system of equations . The postulates are identified (often in the form of parameters), and the implications or predictions are derived by solving the equations . The equations usually describe the average behavior of a large number of interacting molecules or entities a so - call mean - field theory (although variances and other statistical properties can often also be calculated). The solutions are then compared with the experimental measurements the quantitation of the data . If the equations can be solved analytically, all is well and good because this usually indicates that the solution is simple, or at least familiar . If not, numerical solutions are readily provided by such programs as matlab (www.mathworks.com). The predictions of the model (with appropriate parameters) can then be used to quantitatively account for the experimental data . This is the process of curve fitting, which can be used to ask whether the theory actually fits the data (i.e., establish plausibility) and to measure the underlying parameters and their uncertainties . A simulation is a computer - based imitation of a system . Like a mathematical model, a simulation postulates interactions between underlying molecules or entities . Unlike a mathematical model, however, a simulation models individual behavior (rather than mean behavior) and often needs to be repeated a large number of times to obtain an accurate measure of the average behavior . Simulations are especially valuable when modeling small - number systems, which can display highly stochastic behavior . Simulations can be used for establishing the plausibility of a conceptual model, and by changing the parameters, the simulator can experiment with the model to gain insight into how it works . The first is that they often contain many details such as the precise initial conditions or the order of the interactions . In this sense they are often like experimental results, which also depend on many details (e.g., the composition of the buffers). In light of well - documented cases in which the results of simulations depended on these details, it has been argued that a simulation should be treated like an experimental result in the sense that it should be confirmed in different laboratories before being accepted (mitchell, 2009). A second potential limitation of simulation is that although it can establish plausibility of a mechanism, it might provide only limited insight . This is especially the case if the system behavior is unexpected and the elementary interactions give rise to so - called emergent or collective behavior . In this case, the behavior of the simulation may be as mysterious as the biological phenomenon being modeled, even when the simulator can probe the model by varying the model parameters (analogous to performing wet experiments). An alternative approach is to use so - called toy models (or idea models; mitchell, 2009), which, while oversimplifying the system under study, are nevertheless simple enough to solve using a mathematical model, thereby providing insight into mechanism . An example of a toy model is turing's reaction - diffusion mechanism of pattern formation (turing, 1952; gierer et al ., 1972). I do not want the reader to think that simulations are always inferior to mathematical models . Indeed, there are now very good algorithms that have solved many of the technical problems associated with simulations (e.g., the gillespie algorithm; doob, 1945; gillespie, 2007). The physicist freeman dyson recounts attending a conference at which enrico fermi criticized the complexity of dyson's model by quoting the mathematician john von neumann (dyson, 2004): with four parameters i can fit an elephant, and with five i can make him wiggle his trunk . Actually, it is not so easy to model an elephant with four parameters, although we recently achieved it, even making the trunk wiggle with the fifth parameter (mayer et al ., 2010a)! If a model has a small number of premises or parameters that give rise to broad and/or unexpected implications, then we might call it a theory . Evolution the modern evolutionary synthesis is a theory because it makes a relatively small number of postulates yet predicts a very broad range of observations, including the laws of inheritance and models of population genetics . It could be argued that other models, especially those underlying spatial oscillations (e.g., turing patterns) and temporal oscillations (e.g., the cell cycle; see later discussion), rise to the level of theories . Theories and models can be used to establish plausibility, sufficiency, or impossibility . They can also be used to measure . The importance of establishing plausibility is illustrated by a recent example in which a textbook conceptual model was shown not to explain the observations . Myosin cortex was driven by a gradient of tension (bray and white, 1988). However, using experiments (laser cutting) and theory, it was found that cortical flow in the one - cell caenorhabditis elegans embryo is driven not by a gradient in tension but by a gradient of contractility (i.e., motor activity): in the direction of flow, there is no tension gradient, but, counterintuitively, in the transverse direction, there is a tension gradient but no flow (mayer et al ., 2010b)! This example shows the importance of developing a mathematical model (and testing it) to establish the plausibility of a conceptual model . Two examples of the use of theory to establish sufficiency are the action potential and the cell cycle . In a remarkable feat that serves as a foundation for neurobiology, hodgkin and huxley (1952) formulated and solved equations showing that the action potential in nerve cells can be accounted for by the voltage - dependent interactions between sodium and potassium conductances (channels in the modern parlance). The second example concerns the cell division cycle . Following seminal discoveries showing the necessity of phosphorylation for controlling the transitions between phases of the cell cycle, tyson and novak (2011) showed the sufficiency of phosphorylation networks to drive the transitions . In other words, they answered the question, what do you need to build a molecular switch (answer: feedback and cooperativity), and, by doing so, they made the problem of the cell cycle finite . In the process, they made key predictions that were confirmed experimentally and they explained many phenotypes . An excellent example of the use of theory to show impossibility is the prescient work of ken machin (machin, 1958). He proved that the periodic bending motion of cilia and flagella (such as sperm tails) cannot be driven by motor activity localized to the base of the cilium only: if the beating were driven by a whip - like process at the base, the amplitude would die out with a characteristic shape due to the damping from the fluid . This shape is not consistent with the observed beating patterns, whose amplitudes are maintained along the length of the flagellum . Instead, he proposed motors all along the length, with a traveling wave of activity propagating from base to tip . Not bad, considering that the motor dynein had not been discovered (gibbons and rowe, 1965), nor was it known whether the filaments inside the flagellum (the microtubules) slid or contracted (they slide; satir, 1965). Of interest showing impossibility is the most powerful use of mathematical models: it allows hypotheses to be falsified, which is difficult to achieve using simulations . In my work, i have used theory as a tool to measure molecular properties and interactions . For example, we used precise mechanical measurements on hair cells the sensory receptors underlying the sensation of sound and acceleration to estimate the force needed to open a single channel, the displacement associated with the opening of the channel's gate, and the number of channels (howard et al ., 1988). In other work, we used theory to infer the bending stiffness of actin filaments and microtubules from an analysis of their shape fluctuations (gittes et al ., 1993). More recently, we showed how molecular interactions between kinesin motor proteins and microtubules can give rise to length - dependent microtubule depolymerization, which may play a key role in determining the size of organelles such as the mitotic spindle (varga et al ., 2006, 2009; these include the following: morphology: what determines the size, number, and shape of cells and organelles?signaling: how do molecules compute, and what is the role of spatial localization and compartmentalization?scaling: how do we bridge from one scale to another? Are there universal laws? Morphology: what determines the size, number, and shape of cells and organelles? Signaling: how do molecules compute, and what is the role of spatial localization and compartmentalization? Scaling: how do we bridge from one scale to another? Are there universal laws? Especially important is to understand how variability is minimized within a cell to produce reproducible structures and signals, and amplified among cells to enlarge the number of stimuli to which a population of cells can respond . There has been rapid development of single - molecule techniques, making it possible to manipulate individual molecules using optical and magnetic techniques and to visualize their activity inside cells using fluorescence microscopy . In this way it is even possible to sequence single molecules of dna (www.pacificbiosciences.com). At the whole - cell level the developments are as impressive: image processing techniques can be used to segment single cells in tissues to measure shape and activity, their total protein assayed by mass spectrometry, and their genomes sequenced . These studies have revealed remarkable cell - to - cell variability of identical cells . Hand in hand with these developments, we must have theory and models to make sense of the experimental results, as exemplified by gauss, rather than the collection of data for their own sake . I predict that in the future, theory will drive biology as it does physics and engineering.
Dementia as one of the major causes of disability and dependence amongst older people is an important aspect of formal service provision within aged care . Dementia is an umbrella term applied to more than one hundred degenerative brain syndromes which include alzheimer disease, vascular dementia, dementia with lewy bodies, and frontotemporal dementia . These conditions are characterised by cognitive impairment and can result in communication difficulties, loss of memory, problems in performing previous routine tasks, and personality, mood, and behavior changes [1, 2]. The well - being of people with dementia has been linked to the quality of their relationships with their informal carers . In spite of research, both in australia and elsewhere in the world, identifying the significant levels of strain and burden informal carers often experience from the emotional stress and physical burden of caring for a person with dementia the uptake of formal in - home and community care services is lower among the carers of people with dementia than for other conditions associated with ageing [4, 5]. Canadian research suggests that among the explanations for the poor uptake of services is that the informal carers of people with dementia are either unaware of the services which are available or that these services are inaccessible, inconvenient, or too expensive . There are also crucial differences in power, status, and authority between the informal and formal care system that need to be considered in the intersection of the two systems . The policies and practices of the formal care system are conceptualized in rational and decontextualized terms, whereas the expectations and experiences of informal family carers emerge out of a unique set of personal circumstances . This suggests a need for medical and aged care services to consider not only the impact of caring for a person with dementia on their family, but also the family's perspective on formal services [2, 79]. The aim of the current paper is to explore the intersection of informal family caregiving and the formal care systems from the perspective of the primary family carers of people with dementia in an australian context . The study focuses on issues of access to and use of formal services in dementia care from the perspective of the informal family carers . Recent research has found that the decisions made by informal carers on whether to seek or reject formal service provision arise through a complex interplay of social, moral, emotional, and cultural issues . Thus, negotiating the intersection between family carers and those involved in designing and delivering formal aged care services involves social actors who belong to different systems each of which has specific configurations and ideologies . In australia, each year, more than million older people (out of a total population of just under 23 million) receive some form of formal support through the aged care system . Although older australians prefer home - based care to residential care, the greatest proportion of government funding for aged care services continues to be spent on residential aged care services . In june 2010 the distribution of government subsidized aged care places for people aged 70 years and over within the state of queensland (the state where the study reported in this paper was undertaken) was 95.6 per thousand in residential care, 3.9 per thousand in high level community care, and 20.8 per thousand in low level community care . The service provision responsibilities are split both at the level of funding and frontline services for people who are ageing . The australian service system has two streams of community service provision; home and community care services are jointly funded by the national and state governments, whilst home care packages are fully funded by the national government . Within australia, not only the national and state levels of government do provide two different forms of community and dementia care and support, but also the actual provision of services then occurs through a combination of nonprofit and for - profit providers, which, in turn, may be managed at a local, regional, and state level . As a result in the local area there can be multiple providers with whom the carer comes into contact, in some cases providing the same or similar services . This causes considerable confusion for carers due to the complicated and overlapping formal service system . At the frontline of service delivery this is often perceived to have negative impacts due to competition between providers, along with issues related to internal organizational dynamics and interorganizational relationships . In australia there are economic benefits to government in supporting informal carers to continue to care for people with dementia as long as possible, thus reducing admissions to residential care services . In 2010 across australia the cost of residential aged care provision was on average $48,710 per person per year, with government bearing 69% of this cost (or $33,610); in contrast the average annual cost of providing a person with in - home formal aged care was $7,520, with government bearing 92% of the cost (or $6,918 per year); the cost of mixed informal and formal in - home care was on the average of $11,370, with government bearing 35% of the cost of care (or $3,983 per year); and in - home informal care with the person receiving no support from formal aged care services was estimated to cost $10,880 per year, with government bearing no direct cost . The research reported here was undertaken in the australian state of queensland using a qualitative research design incorporating descriptive thematic analysis . This method was chosen as was congruent with the research aims, allowed for an in - depth understanding of the informal caring experience of family carers of people with dementia, and enabled the experience of carers to be presented from their own point of view . Seventeen family carers involved were recruited from across queensland, the third most populous state in australia with a population of 4.6 million people . Selection of participants included in the study was based on the person's experience of caring for a family member with dementia living at home, irrespective of the duration, frequency, or status of the care relationship . Those recruited came from throughout southeast queensland including metropolitan brisbane (the third most populous city in australia), regional centres, and rural communities . All participants were volunteers and recruited through advertisements in local papers, along with distribution of information through two key nongovernment bodies in the state: the alzheimer's association and carers queensland . Eight of the interviews were conducted face to face and nine via were conducted the telephone including all of those with participants living in rural and remote areas of the geographically large state . The sample included two males and fifteen females, a gender balance consistent with the distribution of primary carers of people with dementia in australia . In the study carers were categorized into two groups based on the age of eligibility for the australian aged pension as younger carers where they were under the age of 65 years and as older carers when they were aged 65 years and over at the time of the research interview . The sample includedtwo sons both of whom were aged less than 65 years;five daughters, all aged less than 65 years;one daughter - in - law aged less than 65 years;nine wives, of whom two were under the age of 65 years and seven aged 65 years and over . Two sons both of whom were aged less than 65 years; five daughters, all aged less than 65 years; one daughter - in - law aged less than 65 years; nine wives, of whom two were under the age of 65 years and seven aged 65 years and over . The transcripts were read several times to develop a coding scheme based on thematic analysis of the data . To improve audibility, other members of the research team also reviewed the transcripts and the emergent themes . The first author, who conducted all the interviews, brought to the experience both her background on dementia care as well as on policy and practice development in aged care . That is, within the research process neither the researcher nor the person being interviewed came to the interview untouched by the experience of caring for a person with dementia, albeit from very different perspectives . Other members of the project team provided their own perspective from their work in health services management and sociology of health . Rigour was gained through incorporating three key elements validity, reliability, and verification related to sampling, the position of the researchers, and assessment of whether the objectives of the research were met into the analysis process . The analysis included a constant comparison method with roots in grounded theory along with descriptive thematic analysis, an inductive, or data driven, method that began with the development of a coding matrix based on the questions used in the interviews . The themes were named, defined, described, and refined though a six - stage process which allowed the analysis to become richer in explanation as the process progressed [14, 15]. A subsampling technique, used as a comparative technique, was used to draw out codes by comparing each participant's experience, considered unique in its own right . The clustering of thematic codes to develop higher order themes was a crucial step in the identification of patterns connected to the broader questions under investigation . These higher order themes were then arranged into a conceptual clustering matrix to allow conclusions to be drawn from the data . Through this rigorous process the key findings of the research were schematized to answer the question of how family carers of people with dementia living in the community were successfully managing, continuing, and sustaining informal caring in the present and how they would continue to care in the future . The coding schema was reviewed and corroborated over several face - to - face discussions by three senior academic researchers, who are also coauthors of this paper . The uncertain and unpredictable nature of caring for a family member with dementia was one of the key concerns of the carers, influencing their ability to manage and continue with their caring situation . The carers described the total change in their lives as a result of their caring responsibilities and expressed their desire to continue caring for their family member with dementia but noted that in order to do so they needed access to formal services capable of providing more flexible support that recognized the concerns and issues experienced by carers . From the carer's perspective recognition of the importance of their role was about ensuring timely provision of information and access to formal care early in their caring journey, rather than access to formal care only as a crisis response when their situation deteriorated . The two major themes which emerged were related to the challenges they had experienced in their interaction with medical and aged care services and enablers and impediments at the level of formal community support services . The analysis revealed several distinct subthemes embedded within both the major themes . In relation to interaction with medical and aged care services the sub - themes included delays in the initial diagnosis of dementia, barriers experienced in seeking access services, and a lack of understanding of the needs of informal carers of people with dementia by service providers (see table 1). For enablers and impediments at the level of formal community support services, the participants identified two key characteristics of formal community services which would assist in their ability to balance the effort of caring and their own capacity to continue to care: quality of in - home and day care services and appropriate and accessible opportunities for carer participation in policy formulation and training of staff employed by the aged care and community - based formal services (see table 1). The family carers described the challenges they had experienced in gaining the initial diagnosis and accessing appropriate medical treatment for their family member with dementia, including timely access to appropriate specialist care . Several carers suggested that the local general practitioners familiarity with their family member interfered with the objectivity of the medical assessment impeding early diagnosis . Two of the carers explained in some detail how they felt it was their family member's past medical history, of posttraumatic stress disorder in one case and mental illness in another, which had contributed to the delay in diagnosis . In the first case it had taken seven years from the time the first symptoms of dementia became apparent and were drawn to the attention of the local general practitioner to the eventual diagnosis of dementia . As the wife / carer explained seven years is a long time to wait for a diagnosis . In the second case there had been a three - year delay in diagnosis, resulting in lost opportunities in medical treatment . As that man's wife / carer explained, you need to be on them (referring to drug treatment) early . So, not much use waiting for the doctor to decide that there is a problem, after three years of, of saying it's depression not only some of the family careers had experienced delays in diagnosis and referral to other medical services but also this had then been exacerbated, in several cases, by a poor quality response by the medical professional at the time of the disclosure of the diagnosis . Carers described doctors who failed to take the time to listen or respond to the queries which were raised in ways they felt as insensitive to the needs of both the person with dementia and their own needs as the person's primary carer . The carers also identified the impediments they had experienced in referral to specialist medical services in dementia care . Regardless of where they lived the family carers identified timely and appropriate access to specialist services as a crucial element in their interaction with and confidence of the formal service system . One carer described the delays she and her mother had experienced in accessing appropriate services in terms of age discrimination embedded within the health care system as follows: and so now a geriatrician is taking care of her but such a long - winded process that should have been so much easier . Had it seemed to us that she mattered, but she just didn't seem to matter . So my feeling was well perhaps when you're over 65 or and / or experiencing dementia, that you just drop to the bottom of the pile, i find that incredibly offensive . And so now a geriatrician is taking care of her but such a long - winded process that should have been so much easier . Had it seemed to us that she mattered, but she just didn't seem to matter . So my feeling was well perhaps when you're over 65 or and / or experiencing dementia, that you just drop to the bottom of the pile, i find that incredibly offensive . Several family carers described how they had been left feeling stressed and demoralized when the medical specialist failed to take the time to properly listen to and adequately respond to their questions and concerns, around the implications of changes in the person's behaviour and capabilities . The following description by a wife / carer is indicative of the absence of explanation, advice and support by some medical specialists, the doctor just said, just go home and live out the rest of your life as well as you can . A specialist's dismissal of the carers concerns, can also mean a misdiagnosis as in this description by a daughter / carer: so she was sent to a geriatrician at the base hospital . He was quite good but a little bit arrogant, a little bit condescending, a little bit smug, dismissed dementia and said, no it wasn't dementia, it certainly wasn't alzheimer's and he thought it was probably ms . He was quite good but a little bit arrogant, a little bit condescending, a little bit smug, dismissed dementia and said, no it wasn't dementia, it certainly wasn't alzheimer's and he thought it was probably ms . This misdiagnosis added to the challenges being experienced by this family carer, as the care recipient continued to display symptoms associated with dementia, yet the misdiagnosis meant that the daughter was unable to access appropriate dementia - specific services and support . Fortunately not all medical practitioners consulted by those involved in the current study had acted in this dismissive way, with several of the carers describing medical practitioners who had been very helpful, ensuring both their family member with dementia, and they as the primary carer, understand the medical implications of the dementia diagnosis . Almost all the carers described challenges they had experienced in getting appropriate and timely information about the nonmedical support services available to people with dementia and their carers . Initially this was indicative of the poor intersection between medical and non - medical services in aged care . As one wifecarer explained, my gp is very nice and very good, but he didn't mention where to go or how to access it (non - medical information and support). Because (medical practitioner) is the first port of call, if even if he just had a paper with a code on it that this is alzheimer's society or whatever name, give them a call, that was all that was needed . The carers believed that information on support services was of equal importance as medical assistance in the diagnosis of dementia . Overall what emerges from the interview data was the importance of the doctor's approach in supporting the family carer's capacity to manage the caring situation, especially in planning for future care needs . As explained by one wife / carer the compounding impact of providing informal care is invidious, because you feel duty - bound to keep going, and i think because you've eased into it month by month, year by year you just eased in and you're doing more and more and more and you don't know . Because you feel duty - bound to keep going, and i think because you've eased into it month by month, year by year you just eased in and you're doing more and more and more and you don't know . A number of carers described their frustration that even where they had been provided with information on services in their local area early in their journey as carers this had often been out of date . Carers had also experienced different agencies duplicating the same information, resulting in overlap and confusion, yet still leaving them with gaps in their understanding of the extent of the options potentially available through the formal aged care system . Carers also described the confusion resulting from a dual stream of community service provision between national, state, and local systems which results in multiple providers with which the carer comes into contact, in some cases providing similar or the same type of services . Rather than perceiving the choice of providers as positive, the carers felt that a lot of resources were being wasted on managing an unnecessarily complicated and overlapping service system . Several carers highlighted the negative impacts of competition between providers, along with issues related to internal organizational dynamics and interorganizational relationships . In the present study those family carers with previous experience in engaging with the health and community sectors found that being familiar with the ways support services operated advantaged them in negotiating at the intersection of formal and informal care and support . The lack of familiarity with the organizational logic and work routines that underlies such interactions served to heighten family carers sense that they have no control over the situation they have found themselves in, at a time when they are already stressed and vulnerable . To add to the confusion they found that a single term, such as respite care, was often being applied to differing forms of service provision . For example, in relation to respite care, the same term is used for in - home care, a centre providing day respite, and a block of respite care in a residential facility . Carers also highlighted their difficulties in accessing services soon after diagnosis, with several carers indicating that the services they had contacted appeared to give little priority to the needs of people in the early stages of dementia . In rural areas, in particular, several carers were given the choice of either accessing generic services unsuitable for the needs of their family member with dementia or dementia - specific services which were inappropriate as they were designed around the needs of those with more severe symptoms and behaviours . Other carers highlighted their frustration when they found that the generic community aged care services they had contacted effectively denied access to people with a diagnosis of dementia . Delays experienced in access to some services were also a concern for the carers with long waiting periods not only causing frustration, but also leaving the carer feeling that no one cared for them, whilst they cared for their family member with dementia . Carers described coming into contact with a community aged care system which they felt had become a business, rather than service, a system which cared more about managing finances than meeting the complex needs of people with dementia, along with those of their informal carers . The commodification of services is paradoxical for it also raises carers expectations in relation to the availability of case management as illustrated by the following comment by a daughter / carer: i also believe that if they're providing a seven - day service then there needs to be a contact number for them for all seven days because you can't actually ring them on a saturday morning and say what's happened because there's nobody there . I also believe that if they're providing a seven - day service then there needs to be a contact number for them for all seven days because you can't actually ring them on a saturday morning and say what's happened because there's nobody there . There's no one to answer the phones . A crucial factor that encouraged carers to become actively engaged with formal support services was care workers who were proactive in their contact, rather than relying on the carer to initiate all the interaction with the service provider . In these circumstances the service was more likely to be perceived by the carer as complementing, rather than undermining, their capacity to sustain their caring role . Carers expressed particular concerns about carers effectively having to do their own case management, with the pressure always being on them to initiate contact with services . However, most often the assessment and monitoring processes being used by the formal services were described by the family carers as inflexible and the services as lacking in the ability to respond in a timely way to the constantly changing demands and emergent needs involved in caring for a person with dementia . A failure by assessors in the formal system to adequately consider the needs of the carer is evident in the following description by a daughter / carer: we will only give you x number of minutes in service, we will only do this and we will only do that and it will cost you part of your income and it was just, i thought i honestly suspect that part of this was not meant to be easy to access . We will only give you x number of minutes in service, we will only do this and we will only do that and it will cost you part of your income and it was just, i thought i honestly suspect that part of this was not meant to be easy to access . The carers in the present study believed that their active involvement in the decision - making process would allow for the best interests of the person with dementia to remain paramount in formal care provision . They explained how it was through their involvement in decisions relating to formal services that they were able to develop their confidence and trust in the system increasing the possibility that they would continue to access services . Conversely a complex assessment process, which left carers feeling excluded or peripheral, was identified as an impediment to constructing a comfortable and trusting relationship with service providers . Whilst some carers noted that their role was nominally recognized by services, this was most often only a passive recipient of support, rather than an active participant in the support and care process . The carers emphasized that the formal community aged care system needs to be rebalanced and refocused to ensure that carers are conceived and included as equal partners at the intersection of formal and informal care systems . Carers suggested that this would assist formal care providers to be more responsive and accountable as reflected in the following comment by a daughter / carer: i'd like the care providers to be more accountable to carers and have to explain why they do the things that they do and not just treat their clients like it's a bloody car service or whatever . I think they should be accountable to treat them as people and they just can't have one rule for everybody . I'd like the care providers to be more accountable to carers and have to explain why they do the things that they do and not just treat their clients like it's a bloody car service or whatever . I think they should be accountable to treat them as people and they just can't have one rule for everybody . Carers considered the availability of services to complement their role as informal carers as critical to the future sustainability of their informal care . With many formal services restricted to week - day provision the carers highlighted that services needed to be responsive to the continuous nature of caring by broadening their coverage to seven days a week . The impact of service rationing on the carers own sense of wellbeing and social engagement can be significant as this son / carer explained, i get four hours a month off, which isn't much time to socialize . Other service - related issues experienced by carers included barriers to the transportability of care and support arrangements across the structural boundaries of local, nongovernment, state - based, and national service providers and systems . In some situations, this included those carers who still remained in the workforce and those who would have liked the option to continue working alongside their caring role, but had found it impossible as in the following description by a daughter carer: i had no one to look after mum, so i couldn't go to work, and i do believe that that impacted and i do believe that that's one of the reasons that they fired me . Because i couldn't attend work because i had to look after mum . I had no one to look after mum, so i couldn't go to work, and i do believe that that impacted and i do believe that that's one of the reasons that they fired me . Because i couldn't attend work because i had to look after mum . A number of those receiving formal in - home services in the queensland study described how they felt a need to monitor services when workers unknown to them came into their home . They described circumstances in which in - home respite care assistants were constantly changing, did not turn up when expected, did not demonstrate compassion towards the person with dementia, and did not have good communication skills or demonstrate understanding of the care needs of both the carer and their care recipient . Such issues contributed to the carer's levels of worry and stress as they believed that the vulnerability of the person with dementia and their lack of familiarity with the in - home worker were a risk that needed to be actively managed . The carers described a number of strategies they had used to handle the risks they perceived in using in - home services: staying around the home whilst care was being provided, familiarising themselves with the care system, and most importantly accessing only those services they felt to be reliable and trustworthy . On the same vein the poor physical and interpersonal environment within some out - of - home services encountered by carers deterred them from accessing that service for their family member, particularly when they knew that their family member would not approve the facility . For example, this carer highlights a need to improve both the physical infrastructure of day - care facilities and the education of staff, it is not the facility it is just a chair, and that is hard to send someone to a place for a day where they just sit in a chair . Central to the carers concern was the fact that this situation potentially compromised the safety and security of the person with dementia . In all types of out - of - home environments the carers sought a home - like quality and their ideal was for sufficient respite options to be available so that the most appropriate alternative could be selected, which met the particular needs of their family member with dementia . Creating a trusting relationship with both residential and in - home respite services was factor which had helped the carers feel more confident about dealing with emergencies and sudden changes to care arrangements . Almost half of the carers described the vital role of respite care in allowing them to attend regular commitments which included, but were not limited to, paid work and carer - related support activities (e.g., attending support groups and committee meetings). Respite, in these terms, was not necessarily providing the carer with a break from engagement in instrumental tasks, just time away from the care recipient to complete necessary chores or to earn a living . They also identified a need for services to be responsive to the needs of carers recovering from personal crises, including those that were health - related . It was through consistent, good quality, service provision that the trust and confidence of the carer in formal care arrangements gradually increase . The carers considered consistency in workers providing good quality care and support to be fundamental in meeting the needs of the care recipient and through this in also supporting them to sustain their informal care . Almost half of the carers interviewed already played an active role in providing support for other carers, reflecting the recruitment process for interview participants through the alzheimer's association and carers organizations . Despite this carers also highlighted barriers in participation in consultation processes such as carer forums and carer support groups, including issues related to the timing and location of such events, as in the following comments of this daughter / carer: i got an email the other day and they're actually having a forum in one of the big cities . This is the first time i've ever been asked but the thing is that i think it's at 10:30 in the morning, do you know what i mean? I got an email the other day and they're actually having a forum in one of the big cities . This is the first time i've ever been asked but the thing is that i think it's at 10:30 in the morning, do you know what i mean? Several of the carers highlighted the vital importance of carer input into policy and planning and implementation of formal service provision . These carers felt they had inside knowledge from their caring experience that would be extremely valuable to improving formal services, as expressed by this daughter / carer, it doesn't matter how many university degrees and that people do, they don't know until they're a carer . These carers indicated that a greater investment in making support and training available to ex - carers to provide peer support would bring systemic benefits, including better carer and care - recipient outcomes . They suggested that combining professional expertise and peer support would assist the carers of people with dementia to work through their specific, unique, sensitive, and complex issues . Peer support was identified as important both in helping primary carers adjust to their changing circumstances and in assisting other family members to better understand the impact of dementia on the primary carer . Several carers indicated that they would be willing to contribute to the education or training of formal dementia care workers through sharing their own experience . They also highlighted the staff retention issues within community care services, due to the poor pay and conditions of frontline workers in formal community aged care, as in the following description by a carer / daughter: well to tell you the absolute truth i think that they need to pay them more money . The actual people that come to the door and help these people are on the most terrible wage imaginable . Well to tell you the absolute truth i think that they need to pay them more money . The actual people that come to the door and help these people are on the most terrible wage imaginable . These carers noted how the complex design and implementation of the service delivery system placed additional stress and strain not only on themselves as informal carers but also on frontline workers . At the interface of the two systems the frontline carer and the informal family carer come into contact within a social contract of providing care to the person with dementia in a form which exceeds the reciprocity which usually exists between adults, and therefore the continuity in workers allows for these interrelationships to develop and deepen which can be crucial to the sustainability of informal care . The present study, along with the other recent research focused on the carers experience in dementia care, indicates that the quality of interaction is key to whether the relationships established add to or reduce the burden of care on family carers . There are three key points where changes to policy and practice in the formal care system could improve the capability of informal carers to continue to care . The first occurs when the early symptoms of dementia in the care recipient become apparent and lead to initial contact of the carer with the medical system . At this point information is very much a two - way flow between the informal carer and the formal community care and support system . It needs to be recognized that family members have historical information about the person that, if shared in a timely manner, has the potential to benefit everyone involved . Previous studies both in australia and elsewhere in the western world have found that the general practitioner's role is often vital, not only in the early detection of dementia, but also in the timely referral of the person to specialist services [2123]. The australian institute for health and welfare highlighted in their recent report on dementia care in australia that as there is not a single or simple test that will definitively diagnose dementia the general aim is for the medical assessment process to gather sufficient information about changed behaviours, functional capacity, psychosocial issues, and relevant medical conditions to allow a diagnosis to be made . However, a recent survey of the dementia - related health literacy in the australian and english general practice found that only 22 percent of general practitioners and 15 percent of practice nurses considered their dementia knowledge to be adequate . It has previously been argued that when medical professionals are discussing dementia as a condition issues of concern must be considered from the perspective of both the person with dementia and the primary carer . As mentioned earlier, the hierarchical organization of healthcare services and the esteemed status of health professionals particularly doctors as primary holders of knowledge leads to asymmetrical power relations in clinical encounters . Carers often feel disempowered as they have limited voice in relation to information on services and care on offer for their family member with dementia . There are also acknowledged structural constraints within the australian medical system, with recent research finding that particularly in regional australia general practitioners may delay referring a patient to a specialist for further testing, in part because of more limited access to specialist services . The second critical point occurs when there is a change in support needs as the condition of the person with dementia changes, such as increasing concerns by the carer about the safety of their family member or the emergence of challenging behaviours . This is the point at which access to appropriate respite care may be crucial to the sustainability of informal care . Various approaches that would improve carers engagement with formal community care services, such shifting the focus of formal case management away from making referrals and providing a linking service between services, to a focus by the case manager on organizing personalised and flexible services which not only considered the present issues but also anticipated the future challenges for the carer . This includes consideration of the impact of the demands of caring the lives of people caring for a family member with dementia beyond their caring role to ensure that carers continue to have opportunities for economic and social engagement . A recent inquiry by the australian human rights and equal opportunity commission highlighted the challenges faced by australians seeking to combine work and informal caring responsibilities and identified serious implications for the emotional and economic wellbeing of carers over the long term [26, 27]. In the present study, the stress being experienced by the carers was identified by them as risking their ability to continue to fulfill their informal caring role alongside other responsibilities in their lives . It is important to note that some of the younger carers were combining their role as a primary carer with other family and work commitments, whilst several of the older carers interviewed in the present study did not have access to practical hands - on informal support on which they could readily draw in a crisis . Health economists have conceptualized the burden of informal care in dementia as the putative cost of the time spent by the informal carer on the tasks of care . In their recent meta - review on informal dementia care costa and colleagues assessed the objective burden of informal care in two common dementia - related conditions: firstly alzheimer disease at 55.73 hours per week and secondly parkinsons disease at 15.8 hours per week . They then linked this objective measurement to the subjective evaluation of caregiver burden or strain noting that the more the number of informal hours increases, the more the subjective burden is important . This is most evident where the person with dementia is exhibiting emotional difficulties and/or challenging behaviours [9, 2628]. The assessment of the subjective burden of informal care by formal services needs to better understand the contextual, psychological, and emotional dimensions of care from the perspective of the carer . The gap between apparent need and actual uptake of services points to an important mismatch between the logics operating within organizational systems and the logics within the family environments in which most of dementia care occurs . For example, whilst formal systems tend to base their measurement of need around an abstract and lineal conceptualization of time, for the person with dementia and their informal carer (as well as some frontline staff) the framing of time is enmeshed with social relations and involves processes which cannot be hurried . Other australian researchers have highlighted that the gain that carers experience in receiving formal services is inherently ambiguous, for whilst formal services are providing support to family carers, they can also be undermining their sense of identity and control over their circumstances . A number of those receiving formal in - home services in the present study described how they felt a need to monitor services when workers unknown to them came into their home . In their tasmanian study lloyd and stirling similarly noted that whilst access to formal services can offer physical, psychological, and emotional relief to informal carers it can also cause the carer to experience a threatening loss of mastery over the material and symbolic boundaries of previously private spaces . The third critical point occurs when the burden of informal care is being experienced by the carer as so great that some form of transition is imminent in the care arrangement . As in the other research (e.g., [3, 6, 13]) the carers in the present study were committed to caring for their family member in the community as long as possible, yet also being very aware of the fact that there were limits in their capability to sustain their caring role, particularly as dementia (dependent on the condition) can be associated with progressive decline . They wished to begin to prepare in advance for the point was reached in which the person could no longer be cared for at home . The issues which emerged through the present study, combined with other recent research in australia, suggests that for informal dementia care to become more sustainable it is necessary to ensure that formal service provision is flexible enough to respond to the complexity of the circumstances (family, social, and economic), not only for the person with dementia but also for their informal carer / s . There is increasing recognition within australian aged care funding and policy that the care and support of people with dementia is complex and needs to take the particular needs of carers into consideration . However, on the ground carers continue to be faced with confusing funding arrangements and a complex array of services provided by for - profit as well as non - profit organisations . The present study has reinforced the findings of other reports [3, 26, 27] that in order to improve the sustainability of informal care of people with dementia in the community the formal community care system needs to take a more proactive approach in engaging with informal carers . In order to reduce the burden of care on the primary carers of people with dementia there is a need to ensure the carers perspective is being sought both in the development of policy and within everyday practice . Carers have noted that better services, including respite care, medical (professional and primary) services, and more responsive and appropriate information, and flexible and responsive services are the key considerations in the sustainability of informal care.
The prevalence rate in the general population ranges between 10% and 12%, making this disease one of the most common causes of pain, activity limitation, and restrictions in participation . Knee osteoarthritis (gonarthrosis) is one of the most frequent forms of degenerative osteoarthritis . In knee osteoarthritis, pain is the primary symptom and probably the most important factor related to activity limitation and disability . Although the pain in knee oa is multifactorial, acute exacerbations are usually closely related to synovial inflammation . Various studies performed by using arthroscopy have shown synovitis in painful knees of patients with knee oa . On the basis of these reports, many investigators focused on noninvasive imaging techniques, such as magnetic resonance imaging and diagnostic ultrasonography . The term pes anserinus refers to the conjoined insertion of the sartorius, gracilis, and semitendinosus muscles along the proximal medial aspect of the tibia . These three muscles are primarily flexors of the knee; they also influence internal rotation of the tibia and protect the knee against rotatory and valgus stress . In pes anserine bursitis, the fluid - filled bursa in the conjoined insertion of the three hamstring muscles becomes inflamed and causes pain . Patients with this clinical picture may report vague medial knee pain or may present with tenderness and swelling along the proximal medial tibia . A diagnosis of pes anserine bursitis should be considered when there is spontaneous pain inferomedial to the knee joint and a history of trauma or diabetes . Conditions associated with pes anserine bursitis include degenerative joint disease of the knee, obesity, valgus knee deformity, pes planus, and sporting activities . Pes anserine bursitis affects the quality of life of patients with osteoarthritis, and the treatment methods are different . Treatment includes nonsteroidal anti - inflammatory drugs (nsaids), physiotherapy, and injections of corticosteroid, with highly variable responses; recovery can take 10 days to 36 months . Mesotherapy was introduced 50 years ago by michel pistor, a french physician who used this technique as a novel analgesic therapy for a variety of rheumatologic disorders . Mesotherapy is a minimally invasive technique that consists of subcutaneous injections of drugs and, occasionally, plant extracts, homeopathic agents, or other bioactive substances; for this reason, it has been often considered a form of complementary and alternative medicine rather than a conventional medical therapy . The objective of this type of administration is to modulate the pharmacokinetics of the injected substance and to prolong the pharmacologic effects at a local level . One of the main advantages of mesotherapy is that a local pharmacologic effect can be obtained without the need for high systemic concentrations . Intradermal injections of small amounts of active substance where the injection site corresponds to the area of the pathologic condition for example, in low back pain may provide clinical benefits when other therapies are not available, are not effective, or cannot be used for whatever reason . In addition, intradermal administration of active substances in combination with other systemic therapies can produce synergistic effects, and as a result mesotherapy may have dose - sparing effects . Since its introduction, the use of mesotherapy has been expanded, and therapeutic indications have increased . Although mesotherapy is primarily used for osteoarticular conditions, this technique has recently become popular in cosmetic medicine for treatment of edematous fibrosclerotic panniculopathy and local adiposity . The aim of the current study was to evaluate the effects of mesotherapy with diclofenac for anserine bursitis in knee osteoarthritis . The multicenter study was conducted at the d'annunzio university in chieti, italy, and at an affiliated rehabilitation center in florence . It was approved by the local ethics committee and was performed in accordance with the 1964 declaration of helsinki . One hundred and seventeen patients (59 men and 58 women) age 1856 years (average age, 36 years) were evaluated and treated . All them had anserine bursitis, ascertained clinically and by ultrasonography, in association with grade ii kellgren - lawrence knee osteoarthritis (fig . Inclusion criteria were age between 10 and 60 years, grade ii kellgren - lawrence knee osteoarthritis, re - acutization of pain, and synovial inflammation . Exclusion criteria were pregnancy, known hypersensitivity to products, infiltrative therapy with hyaluronic acid, polynucleotides or corticosteroids in progress, drug abuse or alcohol abuse, significant comorbidities (such as the presence of neurologic abnormalities, concomitant severe rheumatic disease, and systemic abnormalities, such as diabetes), a surgical intervention within 3 months before the study, psychiatric conditions, or psychotherapy or physical therapy within 5 weeks before the study . The patients were randomly divided into two groups (a, mesotherapy; b, control) after giving written consent and agreeing to the possible treatment (local or oral). Group a (60 patients; 30 men and 30 women) received nine sessions of mesotherapy with sodium diclofenac (25 mg/1 ml; akis, ibsa, lugano, switzerland), 1 ml for each session, three times per week . Group b (57 patients; 29 men and 28 women) received 21 oral administrations of sodium diclofenac (50 mg; voltaren, novartis, parsippany, nj), once every day for 3 weeks . All patients reported pain during sleep and after a long period of rest, as well as reduction of pain after about a 200-m walk . All patients were asked to provide standard radiographs (anterior - posterior and lateral) of the lumbosacral tract and knee joints . Primary outcome measures were pain intensity, quantified by using a 10-cm millimetric visual analogue scale (vas), on which the patient is asked to mark the degree of pain intensity, ranging from 0 (absence of pain) to 10 (the worst pain imaginable), along with abilities in activities of daily living (adls), ability to participate in sports, level of pain, symptoms, and quality of life, as assessed by the knee injury and osteoarthritis outcome score (koos). All these assessments, along with ultrasonography, were performed before (t0) and after (t1) the treatment period and at 30 days' (t2) and 90 days' (t3) follow - up in all patients . Differences between mean values before and after the rehabilitation period were tested for significance by using two - way analysis of variance for repeated measures . Graphpad prism (version 6) software (abacus concepts graphpad software, san diego, ca). In group a, the subjective pain levels assessed by vas were 71.05 at t0,20.35 at t1, 3.80.85 at t2, and 4.00.85 at t3 (fig . Disability during sports activity at t1 decreased significantly (p<0.05): t0 score, 7010.7; t1 score, 557.9; t2 score, 608.5; t3 score, 608.7 (fig . Perceived symptoms also were significantly reduced (p<0.05) at t1 and at both follow - up times: t0 score, 71.411.9; t1 score, 46.49.4; t2 score, 509.7; t3, score 53.69.0 (fig . Was seen in the effect of osteoarthritis on quality of life at t1 and at both follow - up times: t0 score, 68.813.2; t1 score, 43.810.9; t2 score, 509.7; t3 score, 509.8 (fig . Disability with regard to adls also decreased significantly (p<0.05) at t1 and at both follow - up times: t0 score, 7214.2; t1 score, 478.7; t2 score, 478.7; t3 score, 509.2 (fig . Finally, pain perception was significantly reduced (p<0.01) at t1 and at both follow - up times: t0 score, 7510.3; t1 score, 30.55.5; t2 score, 365.2; t3 score, 415.8 (fig . Error bars represent the standard deviation . A, group a (mesotherapy treatment); b, group b (oral treatment). Trend of disability during sports activity (knee injury and osteoarthritis outcome score [koos]). Trend of disabilities with regard to activities of daily living (adls) (koos). B, the subjective pain levels assessed by vas were 7.51.85 at t0, 31.10 at t1, and 5 at t2 and t3 (fig . The koos findings were as follows: disability during sports activity did not significantly decrease: t0 score, 7515.8; t1 score, 6515.5; t2 score, 6515.7; t3 score, 7017.2 (fig . 3). A significant reduction occurred (p<0.05) in perceived symptoms at t1: t0 score, 71.413.1; t1 score, 60.78.3; t2 score, 64.38.7; t3 score, 64.39.1 (fig . The effect of osteoarthritis on quality of life decreased significantly (p<0.05) at t1: t0 score, 7513.2; t1 score, 5011.0; t2 score, 68.814.5; t3 score, 68.814.7 (fig . Disability with regard to adls was reduced significantly (p<0.05) at t1: t0 score, 70.612.7; t1 score, 61.79.4; t2 score, 66.210.9; t3 score, 67.612.5 (fig . Finally, a significant reduction (p<0.05) was seen in pain perception at t1 and at both follow - up times: t0 score, 7510.4; t1 score, 52.78.3; t2 score, 55.68.8; t3 score, 58.39.5 (fig . In particular, group a had pain remission after a 120-m walk at t1 and after a 160-m walk at t2 and t3 . Ultrasonography showed a reduction of the hypoechoic area related to anserine bursitis only in group a (ie, the mesotherapy group). The aim of this study was to evaluate the effectiveness of anti - inflammatory drugs administered via mesotherapy in patients with local inflammation in grade ii kellgren - lawrence knee osteoarthritis . Present results showed that the administration of nsaids (diclofenac) via mesotherapy can provide the same therapeutic benefit as that induced by conventional (oral) drug administration in relation to pain after a 3-week treatment period . Indeed, both treatments significantly reduced pain intensity and disability in daily life activity; the effect was maintained up to 3 months (30 days' and 90 days' follow - up) only in the group that received mesotherapy . These results are in accordance with previous studies showing that naproxen and diclofenac administered via mesotherapy were more effective than those given orally . The major finding of this study is the similar effectiveness of mesotherapy and conventional systemic therapy, despite the lower amount of drugs administered to patients undergoing mesotherapy (25 mg with mesotherapy vs 50 mg with oral administration) and the lower short - term risk for adverse reactions in the mesotherapy group (3 weeks). The similar efficacy of mesotherapy and conventional therapy, despite different drug dosages, is difficult to explain . Subcutaneous drug administration results in a very slow drug absorption in comparison with other systemic routes, such as oral and intramuscular . Thus, it could be hypothesized that anti - inflammatory drugs administered via mesotherapy achieve a higher drug concentration in the subcutaneous tissue and exert local effects close to inflammatory cells, sensory fibers, and vascular mediators that orchestrate inflammation and pain . This hypothesis could be confirmed by the evidence of reduction of the anserine bursitis showed by ultrasonography in group a (the mesotherapy group) but not in group b (the oral administration group). Although this study did not measure drug plasma levels after the two routes of administration, it is possible to hypothesize that mesotherapy resulted in a lower systemic bioavailability of drugs, with consequent lower incidence of adverse reactions . This could offer a great therapeutic advantage given the high rates of adverse effects associated with nsaid or corticosteroid use in the elderly population . Although mesotherapeutic techniques used in dermatologic surgery have been associated with many adverse effects at injection sites, including atypical mycobacterial infections, urticaria, lichenoid drug eruptions, and psoriasis, no evidence of local reactions was found in the present study . In conclusion, results of this study indicate that combined administration of conventional nsaids (diclofenac) by mesotherapy is an effective and well - tolerated method for managing anserine bursitis in ii grade knee osteoarthritis in the short term and mid - term compared with oral administration of the same drug with different concentrations . However, future studies could have a larger scale and thus also measure plasma bioavailability of the drug . These observations could be of potential interest for the pharmacologic treatment of anserine bursitis in order to reduce the adverse effects associated with high plasma levels of anti - inflammatory drugs.
Congenital abnormalities (cas) concern all diseases of organs or body parts developed in utero . They can be either isolated localized in one organ or multiple affecting at least two organs grouped into a syndrome, a sequence, or an association . Their prevalence is about 14% of all human fetuses considering all types of abnormalities, that is, major (3%) and minor (11%), or lethal, severe, and benign . Among major cas, congenital heart diseases account for 25%, limb defects for 20%, and nervous system abnormalities for 10% . Moreover, cas represent the first cause of infant mortalities, with an increasing proportion (more than 25%) in both developed and developing countries [1, 3]. In 2002 in the usa, cas caused 21% of infant deaths [4, 5]. In the world, more than 10% of infant mortalities congenital abnormalities can develop at any time after the first month of pregnancy . From conception to birth, the human egg, then the embryo, and the fetus have to adapt, at a molecular and transcriptional level, to various changes in their cellular environment . At conception, this environment depends on the micronutritional status of maternal and paternal germ cells and after conception on maternal nutritional status, metabolism, and lifestyle . Maternal diet is the source of all the essential elements that will serve as basic components, transcriptional factors, growth factors, and messengers for embryological and fetal cells signaling and development . Prevention of cas is defined by individual and public health strategies that can reduce their prevalence . These active strategies include nutritional interventions, prevention of maternal infections and diseases, periconceptional care of sick mothers (epileptic or diabetic), control of professional and environmental exposure to teratogens, and special attention to pregnancies exposed to major health determinants such as obesity, tobacco, alcohol, and drugs . One of the major breakthroughs in ca prevention has been the evidence that periconceptional folate supplementation can reduce the risk of neural tube defects (ntds) [710] and other congenital abnormalities like cardiovascular malformations (cvms), cleft lip and palate, urogenital abnormalities, and limb reductions . It is essential to point out here that ntds preventable through folate supplementation are isolated ntds, and exclude other associated ntds grouped in syndrome, sequence, or association of cas which do not fall within the scope of this paper . Congenital heart and central nervous system abnormalities encompass approximately 50% (resp ., 40 and 10%) of the worldwide infantile deaths attributable to congenital abnormalities [13, 14]. Major congenital anomalies are also a source of high morbidity, distress, and severe physical, psychological, and social handicaps . Teratology, the science of the precise etiologies of ca, defines these causes as unknown in 5060% of cases . The other etiologies are epigenetic and multifactorial in 2025% of cases, chromosomic or genetic with a single gene mutation in almost 15% of cases, and epigenetic, acquired, and monofactorial under the influence of environmental factors (such as maternal sickness, infections, medications, ionizing radiations, and alcohol) in about 10% of cases (figure 1). Clinical studies have revealed that a specific teratogen can induce various malformations, or none, depending on the timing of exposure of the developing embryo . Thereby, each organ or system displays a critical, yet brief, window, considered as a phase of susceptibility to environmental teratogens . It is commonly known that the earlier the exposure, the more severe the abnormalities, which can even lead to death of the embryo during the first month postconceptionally . Most deleterious teratogens produce nonspecific congenital abnormalities such as general dysmorphic features, intrauterine demise, or intrauterine growth restriction, as well as specific ca, which can characterize a particular agent . For example, spina bifida occurrence is increased with three principal maternal risk factors and still exhibits the same clinical aspect: maternal valproic acid intake, insulin - dependent diabetes, and folate deficiency . Improved comprehension of etiopathogenesis has led to the emerging evidence that equilibrating and optimizing maternal dietary intake can reduce the incidence of ca . Influence of nutrition on fetal development has been repeatedly proven, at the molecular as well as the clinical level . Very recently, pilot data from a randomized double blind controlled trial showed that periconceptional maternal micronutrient supplementation affected fetal genome methylation patterns in dna samples drawn from cord blood . Folate, or vitamin b9, is most abundantly found in dark green leafy vegetables, but also in orange juice, legumes (e.g., black beans and kidney beans), nuts, asparagus, and strawberries . With the exception of liver, folic acid is the synthetic form of folate and is usually more bioavailable than natural food folate . Due to its lower bioavailability from natural foods, folates are essential for the synthesis of thymidylate and purines, precursors required for de novo dna synthesis and hence, cell division . Folate coenzymes are also implicated in amino acid metabolism (homocystein) and methylation . In order to be stored intracellularly, folate ought to be metabolized into tetrahydrofolate (thf) by methionine synthase, a b12-dependent enzyme . In humans, the association of ca and folate deficiency began to be acknowledged in the 1950s, when methotrexate was widely used for abortions . Moreover, methotrexate and aminopterin, both folic acid antagonists, were being used for the treatment of psoriasis and certain cancers in pregnant women, which resulted in ca, thus starting to reveal an association . Additional research into neural tube defects and their etiologies was further facilitated by the advances in genetics and nonmendelian complex diseases, paving the way for the study of metabolism and transport of folate - homocysteine as potential risk factors for spina bifida . Since then, folic acid supplementation has remained one of the few interventions, if not the only, that can prevent major ca in the human fetus . During pregnancy, folate requirements increase to accommodate embryonic and fetal development and maternal tissue growth . While folate is actively transported to the fetus as demonstrated by higher cord blood folate concentrations relative to maternal blood, maternal serum and rbc concentrations of folate decline for several reasons [1924]: increased demand, dilution secondary to increased intravascular volume, increased folate catabolism and clearance, decreased absorption, and inadequate intake [19, 22]. Folate deficiency is known to lead to maternal megaloblastic anemia, which may be fatal if left untreated . Addressing folate deficiency as it relates to ntds occurrence or recurrence has been the subject of considerable study . The evidence in public health that daily folic acid supplementation (alone or in combination with other vitamins and minerals) has a significant protective effect in preventing ntds, that is, anencephaly and spina bifida as well as cardiovascular malformations is now overwhelming . There is also a significant reduction in risk of recurrence, while prevention of other birth defects (cleft palate cleft lip) and or miscarriages was not proven to be statistically significant . The controversy about the potential role played by folic acid supplementation in the rising colon cancer rates should no longer be defended, as the majority of the evidence available is reassuring . The current identified maternal risk factors for ntds include four established factors: personal or familial past history of ntd (relative risk rr of 30), maternal diabetes (rr, 210), certain antiseizure medications (carbamazepine and valproic acid, with rr of 10 to 20), and maternal folate deficiency (rr, 28) (table 1 and figure 2). Maternal factors such as obesity, hyperthermia, race, ethnicity, smoking, alcohol abuse, malabsorption, intestinal disease, and liver or renal failure, can also contribute to genesis of ntds either directly or indirectly by folate deficiency [25, 28]. Low folate intake, in addition to inadequate absorption of food folate and further loss through cooking practices, leaves the majority of women of reproductive age deficient in folates . As closure of the developing neural tube occurs by the 28th postconceptional day, that is, the 42nd gestational day, before the majority of women are aware of their pregnancy, this precludes the efficacy of folic acid given after the diagnosis of pregnancy . Based on current evidence, it is recommended that all women of childbearing age receive 0.4 mg (400 g) of folic acid daily periconceptionally (1 month before and 2 months after). Women at high risk for ntds, that is, women with previous ntd - affected pregnancy, obesity (bmi> 30), diabetes, and epilepsy, should receive 4 to 5 mg of folic acid daily preconceptionally, starting at least one month before conception and continuing throughout the first trimester of pregnancy . The recommended dose of 4 mg / d was chosen for a medical research council trial that resulted in a 72% reduction in ntd recurrence [16, 29]. The adequate blood folate concentration and minimum supplemental dose shown to be effective for the prevention of ntds are not precisely known . The only randomized controlled trial (rct) showing reduction in occurrence of ntds used an 800 g / d dose, whereas other intervention trials studying occurrence or case control studies of recurrence used a 400 g / d dose . All of the studies demonstrated significant reduction of ntds [8, 10, 30]. However, the ntd risk reduction with higher blood folate concentrations is well documented, as is the enhancement of folate status with the combined consumption of folic acid supplements or fortified foods and a healthy diet containing natural folate [3133]. Proper evidence on folate dose remains limited . For instance, the precise red blood cell folate concentration of 906 nmol / l was demonstrated to be related to a lowest risk of ntds in the offspring . This concentration was not reached within four weeks of the currently recommended supplementation, according to daly et al . . However, brmswig et al . Were able to reach that same target level within four weeks of supplementation with a daily intake of 800 g / d folic acid . These results suggest the need for the reevaluation of the current dosage recommendation of folic acid supplementation with respect to ntd prevention . Although the preventive efficacy of ntds by folic acid - containing multivitamins (mv) or folic acid alone has been well established and demonstrated to be better than any other ca prevention, the available data also supports the essential role of folic acid for normal fetal cardiac development during early embryogenesis . The combination of the results of two hungarian intervention trials [35, 36] (or with 95% ci: 0.57, 0.390.85) showed a 43% risk reduction of cardiovascular malformations after mv supplementation . Two more recent population - based observational studies demonstrated a significant reduction in the rates of cvm with folic acid intake [37, 38]. Furthermore, a significant reduction in the birth prevalence of severe cvms was reported in quebec, canada, after folic acid fortification of grain products . Another canadian study, a systematic review and meta - analysis by goh et al ., concluded that maternal consumption of mv was associated with decreased risk for several congenital anomalies (or 0.78, 95% ci 0.670.92 in case control studies and or 0.61, 95% ci 0.400.92 in cohort and randomized controlled studies for cardiovascular defects). In conclusion, the available evidence concerning cvms shows that any public health action of ca prevention with periconceptional mv or folic acid supplementation should necessarily take into consideration cvms, with regard not only to demonstrated efficacy but also to the more elevated prevalence of cvm as compared to ntd or other defects, and thus to the superior absolute number of preventable cases of cvm per 100,000 births . This should be particularly true in countries with a low ntd prevalence, and a low ntd: cvm ratio, such as the usa . Teratogenicity may exist at the conceptional level as well at the preconceptional level, thus affecting both maternal and paternal gametes . A recent proven example, although it has not been fully elucidated, is the effect of paternal exposure to dioxins . A statistically significant causal link has been demonstrated between dioxin exposure and spermatozoid folate deficiency leading to spina bifida [4143]. The media term dioxins is often used for a family of structurally and chemically related products . Dioxins are mainly unwanted byproducts of a wide range of industrial processes . In terms of dioxin release into the environment, uncontrolled waste incinerators (solid waste and hospital waste) the highest levels of these compounds are found in some soils, sediments, and food, especially dairy products, meat, fish, and poultry . Once the human body has absorbed dioxins, they persist for a long time because of their chemical stability and their ability to accumulate in fat tissue . After preconceptional exposure to dioxins, the risk of mutations in spermatozoids is significantly increased, leading to an increased risk of spina bifida [4143] through mechanisms involving folate deficiency . In animal models, the teratogenic and mutagenic effect of dioxin has been extensively proven, while light had just started to be shed on the mechanism by which human paternal dioxin exposure can lead to congenital abnormalities . The causative and statistically significant association between the dioxin containing agent orange, which was used in the vietnam war, and spina bifida, is irrefutable [4143]. Precise biological mechanisms of exposure to dioxin are epigenetic, based on activation of the ahr / arnt (aryl hydrocarbon receptor / aryl hydrocarbon receptor nuclear translocator) complex of spermatogenesis, leading to folate deficiency as the cause of ntd phenotype [44, 45]. This complex is widely distributed, but is particularly abundant in the human testicle, which renders it one of the most sensitive organs to dioxins . This effect is mediated by a downregulation of rfc 1 (reduced folate carrier 1) gene expression and reduced carrier protein levels . This downregulation by tcdd was shown to be time- and dose - dependent in rat livers and resulted in functional folate deficiency in male and female cells and tissues, including germ cells . In summary, dioxins are diffusely distributed in the environment and tend to accumulate along the food chain . Chronic consumption of contaminated food can lead to deregulation of genetic mechanisms implicated in folate homeostasis . Consequently, widespread folate deficiency in men and women, generally due to inadequate consumption of alimentary folates, can be increased . The final consequences are ntds in fetuses born to mothers, but also fathers, deficient in intracellular folate concentration in germ cells . Paternal folate deficiency could be one of the factors explaining the incomplete success of recommended folate supplementation to prevent ntds . Vitamin b12 (cobalamin) is a pivotal cofactor for key enzyme reactions including the generation of methionine and tetrahydrofolate . This vitamin is found almost exclusively in foods of animal origin (meats, dairy products). Although inadequate vitamin b12 status is thought to be limited to the aging population, it has been found with a relatively high prevalence in women of reproductive age with restricted consumption of animal - based food, an increasingly popular dietary trend, and in pregnant women who are more likely to be deficient than nonpregnant women . Ray and blom identified 17 ntd case - control studies related to b12 status, with an overall reported trend towards lower mean b12 concentration in mothers with ntd - affected pregnancies . The two largest positive studies conducted after the introduction of folate fortification in the united states and canada [52, 53] showed that the risk of ntds was inversely proportional to the measured serum concentrations of vitamin b12 (holotranscobalamin). Furthermore, in ireland, risk of ntds was strongly positively correlated to low b12 status in a population not exposed to folic acid fortification or supplements [54, 55]. Uncovered a fivefold increase in ntd risk for women in the lower quartile of associated folate and b12 deficiencies, compared to half this rate for only folate deficiency, thus demonstrating the synergistic actions of both vitamins . More recently, zhang et al . And molloy et al . Reported higher ntd risk in mothers within the lowest quartile for b12 concentrations, with an approximate three - fold increase . Both kirke and zhang demonstrated that b12 and folate were two independent risk factors . In 2009, the national institutes of health validated the results of molloy et al ., and concluded that improving b12 status in women of childbearing age would prevent ntds . The recommended daily vitamin b12 allowance for pregnant women caregivers should be aware that high levels of folic acid intake (tolerable upper intake level from fortified foods or supplements is 1000 g / d for adults), can mask vitamin b12 deficiency, resulting in permanent nerve damage, especially in elderly individuals . As previously mentioned, the etiology of ca is multifactorial, and most factors are still unknown . Toxic chemicals are detected everywhere, in the dietary products of the general population, in the inhaled air, and even in neonates' cord blood . According to a who report in 2010, the top three most common groups of etiologies for ca in developed countries are epigenetic: maternal diseases (diabetes and hyperthermia), pathologic maternal deficiencies (folate and iodine deficiency), and exposure to teratogens (medications, drugs including tobacco and alcohol, environmental chemicals like pesticides, and ionizing radiations). The unfolding of new associations between malnutrition and diseases has made it clear that women in their reproductive years have multiple complex nutritional deficiencies; this might also be true for the general population . Recent studies showed that prenatal multimicronutrient supplementation was associated with a significantly reduced risk of low birth weight when compared with iron - folic acid supplementation [62, 63]. Moreover, chen et al . Proved that periconceptional multivitamin supplementation containing folic acid two months before conception and until completion of the second month containing folic acid can prevent the occurrence of ntds . They can be due to digestive malabsorption, but also to excessive cooking of fresh food that destroys most of the vitamins, and to widespread consumption of industrial food products . Epidemiological studies, both observational and interventional, have all been consistent with a 50 to 70% protective effect of adequate consumption of folates on ntds . Since strategies to modify women's dietary habits and vitamin use have achieved little progress and since about half of all the pregnancies are unplanned, maternal supplementation alone cannot be an effective approach . Only maintenance of optimal nutritional status throughout the reproductive years will help ensure normal fetal development . Starting in north america, fortification of food with folic acid has made folic acid accessible to all men and women of childbearing age without necessitating behavioral change and has proven to be both efficient and more homogeneous . An additional rationale for fortification is that, in contrast with food folate, bioavailability of folic acid from fortified food is 85% (it is a 100% from vitamin supplement). In 1992, the us public health service (usphs) recommended that all women in their reproductive years consume 400 g of folic acid daily for prevention of ntds . By 1998, and following food and drug administration (fda) regulations, all standardized enriched cereal grain products sold in the united states included 140 g folic acid/100 g. folic acid was also added to breakfast cereals, corn grits, infant formulas, medical foods, and foods for special dietary use . In 2009, the us preventive services task force published updated grade a recommendations reinforcing these guidelines . By 2007, over 50 countries had implemented their own folic acid flour fortification programs, including canada, costa rica, chile, australia, new zealand, south africa, and some middle eastern countries . In the usa, the national birth defects prevention network reported a 36% decrease in the prevalence of ntds, from 10.8 per 10,000 population during 1995 - 1996 to 6.9 at the end of 2006 . In europe, eurocat registries reported a 10% decrease, from 10.5 per 10,000 in 2004 to 9.4 in 2008 . A greater decline in ntds was predicted, raising the question of what additional measures should be undertaken . According to the cdc 2010 report, disparities still exist among diverse ethnicities, as well as on a global worldwide basis . As current fortification programs only prevent about 9% of total annual cases of ntds, an international public health solution is to expand the number of countries with mandatory fortification programs that have the potential to safely diminish the fraction of folic acid - preventable ntds . However, while the abundant literature depicts mandatory fortification programs as massive public health success stories, one study found that neither periconceptional supplementation nor dietary folic acid intake reduced the risk of ntds, including spina bifida . Moreover, another confirmed the protective effect of dietary folic acid alone, regardless of supplementation status, which did not appear to offer further benefit in reducing the risk of spina - bifida - affected pregnancies, even among women with very low dietary folic acid consumption . It is worth mentioning here that these were case - control studies that relied on self - reported maternal questionnaires, which raises the question of reporting accuracy, in the era of global awareness of the protective effect of folic acid . Furthermore, despite folic acid fortification and maternal supplementation, the incidence of ntds has stabilized in the united states and many other countries . All this knowledge highlights the need for paired management strategy: prevention by folic acid and vitamin b12 maternal and paternal supplementations associated with a decreased exposure to the multiple risk factors and treatment such as fetal surgery . The first prenatal myelomeningocele (mmc) repair was performed in 1994 by bruner and tulipan endoscopically on 4 cases . Due to premature labor and birth, this technique was considered dangerous and unsatisfactory and was abandoned . In 1997, the first cases of open mmc repair by hysterotomy were realized [80, 81]. Very recently, adzick et al . Proved, in the management of myelomeningocele study (moms), the efficacy of fetal mmc surgery as compared to standard postnatal repair . Prenatal surgery resulted in significant reduction of hindbrain herniation (chiari ii malformation), a reduction in need for shunting to relieve hydrocephalus, as well as improvements in motor function and mental development at 30 months (table 2) [50, 82]. The etiologies of congenital abnormalities lie in epigenetics in the vast majority of cases . They are multifactorial, maternal as well as paternal, but universally associated with environmental teratogens . Future research and multicentric, large - scale trials should be directed to epigenetic profiling of congenital diseases, including neural tube defects . Scientific as well as political effort is mandatory in order to implement global preventive public health strategies using fortification and supplementation.
Malignant peripheral nerve sheath tumors (mpnst), which are comprised of 5 to 10% of all soft - tissue sarcoma, often occur in the near proximal extremity at a nerve trunk such as sciatic nerve, brachial plexus, or sacral plexus . They have a distinct association with the neurofibromatosis type 1 and occur in 3 - 15% of nf1 patients and a high incidence of tumor is related to the mutation of the nf1 gene9). On the other hand, a primary malignant intracerebral nerve sheath tumor (minst) is quite rare, with only 8 cases1,2,4,10,12,13,15,16) documented in english - language literature . A 13-year - old boy was hospitalized due to the headache on the right side of his head, which began 2 weeks ago and the vomiting, which began a week ago . There was no evidence of neurofibromatosis such as neurofibroma, caf au lait spot, lisch nodule, optic glioma, distinctive bony lesion (dysplasia of the sphenoid bone or long bone cortex) and freckle in the groin or axilla with no family history of neurofibromatosis . Brain magnetic resonance imaging (mri) revealed about 6.7 cm diameter, a well defined highly enhancing mass in the right frontal lobe (fig . 1). The preoperative differential diagnoses were glioblastoma multiforme, anaplastic astrocytoma, and malignant meningioma . Macroscopically, it was a well - circumscribed yellowish nodular round mass having gelatinous surface without dural attachment (fig . It was intra - parenchymal tumor and had a distinct cleavage plane in the superficial brain parenchyma, but not in the deeper portion . Microscopically, the tumor was highly cellular and mainly consisted of spindle cells in interlacing and interwoven fascicles . Each tumor cell had a fusiform nucleus and aligned in a convoluted form with a moderate degree of mitotic activity and a high nuclear - to - cytoplasmic ratio . 3), but negative for glial fibrillary acidic protein (gfap), cd34, synaptophysin and desmin . These histological and immunohistochemical findings supported the diagnosis of a primary minst . Thirty times of fractionated whole - brain radiation were performed with 5760 cgy over the period of 6 weeks after surgery . Brain mri follow - up examination was performed every six months, but there was no evidence of tumor recurrence . Brain mri at 50 months after his first surgery revealed a 1.01.0 cm of well - defined enhancing mass in the right frontal convexity and leptomeningeal enhancement in the right frontal area (fig . The histopathological examination showed a primary minst, which was consistent with the histopathological results from the first surgery . The brain mri at four months after second surgery revealed a tumor of 1.00.5 cm in size in the right anterior frontal convexity and pachymeningeal enhancement in the right frontal area (fig . As the patient's guardian wanted to treat the patient in another hospital, so the patient was transferred to another hospital . As the tumor was repeatedly relapsed despite the gtr which was performed twice before, radiation therapy and chemotherapy were performed according to the korean society for pediatric neuro - oncology (kspno)-s081 protocol, which can be used for a high - grade brain tumor13). Thirty times of fractionated radiation were carried out with 6000 cgy after his third surgery and 4 times of combination chemotherapy with vincristine (1.5 mg / m iv push, day 0, 7), etoposide (75 mg / m in normal saline dose 2.5 times iv over 2 hours, day 0 - 4), carboplatin (300 mg / m in normal saline 125 ml / m iv over 1 hour, day 0, 1), and ifosfamide (1500 mg / m in dextrose water 200 ml / m with mesna (300 mg / m iv over 1 hour, day 0 - 4) were applied . The irregular enhancing lesion (3.04.0 cm) was found in a brain mri at six months after the third surgery (fig . Was found to be smaller than the previous one in the brain mri at thirteen months after the third surgery, this lesion is considered to be the result of radiation injury rather than tumor recurrence (fig . 5c). He is still alive at 77 months after his first surgery and complaining of mild headache and dizziness, but there are no other neurological abnormalities . A 13-year - old boy was hospitalized due to the headache on the right side of his head, which began 2 weeks ago and the vomiting, which began a week ago . There was no evidence of neurofibromatosis such as neurofibroma, caf au lait spot, lisch nodule, optic glioma, distinctive bony lesion (dysplasia of the sphenoid bone or long bone cortex) and freckle in the groin or axilla with no family history of neurofibromatosis . Brain magnetic resonance imaging (mri) revealed about 6.7 cm diameter, a well defined highly enhancing mass in the right frontal lobe (fig . 1). The preoperative differential diagnoses were glioblastoma multiforme, anaplastic astrocytoma, and malignant meningioma . Macroscopically, it was a well - circumscribed yellowish nodular round mass having gelatinous surface without dural attachment (fig . It was intra - parenchymal tumor and had a distinct cleavage plane in the superficial brain parenchyma, but not in the deeper portion . Microscopically, the tumor was highly cellular and mainly consisted of spindle cells in interlacing and interwoven fascicles . Each tumor cell had a fusiform nucleus and aligned in a convoluted form with a moderate degree of mitotic activity and a high nuclear - to - cytoplasmic ratio . 3), but negative for glial fibrillary acidic protein (gfap), cd34, synaptophysin and desmin . These histological and immunohistochemical findings supported the diagnosis of a primary minst . Thirty times of fractionated whole - brain radiation were performed with 5760 cgy over the period of 6 weeks after surgery . Brain mri follow - up examination was performed every six months, but there was no evidence of tumor recurrence . Brain mri at 50 months after his first surgery revealed a 1.01.0 cm of well - defined enhancing mass in the right frontal convexity and leptomeningeal enhancement in the right frontal area (fig . The histopathological examination showed a primary minst, which was consistent with the histopathological results from the first surgery . The brain mri at four months after second surgery revealed a tumor of 1.00.5 cm in size in the right anterior frontal convexity and pachymeningeal enhancement in the right frontal area (fig . As the patient's guardian wanted to treat the patient in another hospital, so the patient was transferred to another hospital . As the tumor was repeatedly relapsed despite the gtr which was performed twice before, radiation therapy and chemotherapy were performed according to the korean society for pediatric neuro - oncology (kspno)-s081 protocol, which can be used for a high - grade brain tumor13). Thirty times of fractionated radiation were carried out with 6000 cgy after his third surgery and 4 times of combination chemotherapy with vincristine (1.5 mg / m iv push, day 0, 7), etoposide (75 mg / m in normal saline dose 2.5 times iv over 2 hours, day 0 - 4), carboplatin (300 mg / m in normal saline 125 ml / m iv over 1 hour, day 0, 1), and ifosfamide (1500 mg / m in dextrose water 200 ml / m with mesna (300 mg / m iv over 1 hour, day 0 - 4) were applied . The irregular enhancing lesion (3.04.0 cm) was found in a brain mri at six months after the third surgery (fig . Was found to be smaller than the previous one in the brain mri at thirteen months after the third surgery, this lesion is considered to be the result of radiation injury rather than tumor recurrence (fig . He is still alive at 77 months after his first surgery and complaining of mild headache and dizziness, but there are no other neurological abnormalities . The primary minst is termed as a result of their histological and immunohistochemical similarities to mpnst but is distinguished by their intracerebral location1). Minst is a preferable term to indicate malignant intracerebral schwannoma or neurofibrosarcoma, since these tumors have the appearance of any nerve sheath cells, including schwann cells, perineural fibroblasts, or fibroblasts8,10). Neither our case nor the other previously - published case reports could make an accurate diagnosis before pathologic findings . In imaging studies such as brain computed tomography and mri, it is difficult to discriminate between minst and high grade glioma, because those tumors were intra - axial, homogeneously enhancing and had surrounding edema11). It is difficult to make an accurate diagnosis of minst by looking ordinary hematoxylin and eosin - stained sections alone . The differential diagnosis of these tumors includes malignant melanomas with schwann - type differentiation, mixed gliomas with mesenchymal differentiation, intracerebral rhabomyosarcomas, desmoplastic infantile ganglioma, intracerebral meningioma, and solitary fibrous tumor2,16). However, immunohistochemical analysis has special value both in verifying nerve sheath derivation and malignancy16). Positive s-100 protein may help in differentiating the nerve sheath tumors from the other soft tissue tumors6). The absence of gfap expression in tumor cells ruled out desmoplastic astrocytoma, gliofibroma, and gliosarcoma, whereas the absence of synaptophysin and neurofilament protein excluded desmoplastic ganglioglioma12). In the reported cases, the prognosis of minst is poor2). According to the data in table 1, survival time was longer when the tumor recurrence was slower . The recurrence free survival after the initial resection appears an important predictor of the overall survival12). In cases of bruner et al.4), barnard et al.1), and this case which reported long - term survival, the mitotic activity was rare or moderate rather than high . It seems that if the mitotic activity is low, the recurrence rate is also low and thus the survival time will increase, although we could not take into account of various factors due to insufficient data caused by few number of reports have been produced to date . A longer survival time was reported when total resection rather than subtotal resection was carried out3,7,16). It was considered that radiotherapy contributed to playing a limited role in the management of tumor in the early literature14,15). However, in the recent literature, radiotherapy showed possible effects on the local control and recurrence2,5), but little on the overall survival in patients with minst1,16). In case of beauchesne et al.2), systemic chemotherapy with doxorubicin (3 courses every month) was prescribed, with mild benefit for the patient . However, because of the rarity of this tumor type, there are still few reports published and little available information about the chemotherapy8). In this case, the kspno - so81 protocol was applied, which is one of protocols to treat a pediatric high grade brain tumor such as medulloblastoma and primitive neuroectodermal tumor . Although appropriate regimen of radiation therapy and chemotherapy are not determined yet2,5,15), it is considered to be useful for the long - term survival of the patient to carry out a surgery, adjuvant radiation, and chemotherapy, as shown in this case . Different from the previous cases, this is the first case that had no evidence of tumor recurrence for 50 months after first surgery, and showed long - term survival for more than 77 months without showing no evidence of tumor recurrence . Although a definitive therapeutic regimen for minst has not yet been established due to the limited number of reported cases, a gtr followed by adjuvant radiotherapy and chemotherapy will be recommended to patients of minst.
Aegypti was maintained in the laboratory of chemical ecology of vector insects of the federal university of minas gerais from 2007 . The breeding room was kept at a temperature of 27 1c, 75 - 80% relative humidity (rh) and a 12l:12d photoperiod (eiras & jepson 1991). The larvae were kept in plastic tubs containing water (approximately 5 cm deep) and fed reptile food (reptolife, alcon, brazil) until they reached the pupa stage . The pupae were collected daily and transferred to adult screened cages (bugdorm-1, mega view science education services, taiwan) (30 30 30 cm) and fed 10% sucrose solution . A selection cage was used to separate the gravid females that were used in the assays . Blood meal was prepared using chicken blood (gallus domesticus), which was obtained from a slaughterhouse, including the anticoagulant heparin (gomes et al . . Aegyptifemales (f6 and f7) of five-10 days of chronological age and three days after blood meal (gomes et al . Experimental area - the tests were performed in laboratory and semi - field conditions . In laboratory conditions, four transparent acrylic boxes (150 50 41 cm) were kept in a room with monitored temperature and humidity conditions (27 3c, 60 - 90% rh) and a photoperiod of 12l:12d (eiras & jepson 1991). The semi - field tests were performed in an experimental area of 14.0 7.0 3.5 m, where eight cages (2.5 2.5 2.0 m each) were installed (roque & eiras 2008). The temperature, rh and the photoperiod ranged according to the external environment and were monitored with a thermo - hygrometer . The tests were considered valid only when the temperature reached a minimum of 25c (roque & eiras 2008). Bioassays - the tests evaluating the pattern of distribution of eggs by ae . The females were evaluated in laboratory conditions in experiment 1 and in semi - field conditions in experiment 2 . Experiment 1 (laboratory) was performed in the four acrylic boxes in which four different densities of breeding sites (2, 4, 8 and 16) were simulated . In each box, a single gravid female of ae . Aegypti was released and allowed to remain for 96 h in the interior of the box . The breeding sites were ovitraps that were set with 300 ml of tap water and a rectangular wooden paddle (12 cm 3 cm) (fay & perry 1965, fay & eliason 1966). For each density level, 12 repetitions of the experiment were performed . 1). Sugar solution (10%) absorbed into cotton was provided as food to the insects during the experimental processes . 1:representation of the distribution of the different densities (2, 4, 8 and 16 breeding sites) and the positions of the breeding sites in the interior of the acrylic boxes . In the centre, after 96 h, the paddle and the water that were present in each trap were collected, labelled and taken to the laboratory . To verify the distribution of eggs at each breeding site, the number of eggs that were present in the water and in the paddle of each ovitrap was counted using a manual counter and a stereoscopic microscope (20). Experiment 2 (semi - field) was conducted in the interior of the four fabric cages (2.5 2.5 2.0 m) (roque & eiras 2008). The same densities (2, 4, 8 and 16 ovitraps) that were evaluated in the laboratory conditions were evaluated in this environment . For each density the positions of the breeding sites were kept constant and the same methodology as used in experiment 1 was followed . However, to avoid visual effects of the treatment itself, each cage received all 16 of the ovitraps, but only the test traps (densities 2, 4, 8 or 16) received water (fig . 2). Fig . 2:diagram of the distribution of the ovitrap with and without water of each treatment / density of breeding sites . Statistical analyses - for statistical analyses, the specific locations of egg deposition (water and paddle) were called habitats and the local of performance of the experiment (laboratory or semi - field) was called environment . To compare the number of eggs that were deposited in each environment / density, data were submitted to a normality test at a significance level of 95% . When the distributions were not in accordance with the criteria of normality, medians were compared using the kruskal - wallis test . The proportions of eggs that were deposited on water by each single female (n eggs on the water / n total eggs) at the different densities of breeding sites both in laboratory and semi - field conditions were compared by two - factors anova . Before analyses, the data were transformed in arcsin(sqrt) to normalise the distribution and stabilise the variances . The number of colonised breeding sites in relation to the amount of available ones, both in laboratory and semi - field conditions, was compared by the kruskal - wallis test . By investigating the female groups above and below the regression lines, we obtained two groups: those that colonise various breeding sites and those that colonise few sites . We compared these two groups by the mann - whitney u test to determine whether there was any difference between the groups with higher and lower frequencies of colonised breeding sites and the proportion of eggs that were deposited in the preferential breeding site . The r program (available from: r-project.org) environment (laboratory or semi - field) of the experiment and egg deposition - the oviposition behaviour of ae . Aegypti was maintained in both environments . Under laboratory conditions, the mean number [standard deviation (sd)] of eggs that were laid by one ae . Aegyptiper repetition was 73.9 (25.64), ranging between 40 - 142 eggs . Similarly, under semi - field conditions, the mean number (sd) of eggs that were laid by oneae . Aegypti per repetition was 72.2 (20.57), ranging between 41 - 123 eggs . On average, females in both the experimental environments distributed the same number of eggs and colonised the same number of breeding sites (anova p> 0.05). Therefore, these oviposition behaviours do not seem to be influenced by changes in the tested environments . Oviposition on the water - the tested females exhibited a strong tendency to deposit their eggs on the water of the ovitrap . The mean proportion of eggs that were laid on the water surface tended to be higher than that on the paddle when the number of available breeding sites was greater (fig . Habitat received significantly more eggs in semi - field than in laboratory conditions (anova p <0.05) (fig . 3:box plot of the proportion of eggs on the water in different density of oviposition breeding sites . 4:box plot of the number of eggs laid on water and paddle in semi - field and laboratory conditions . Breeding sites colonised in relation to available ones - the availability of breeding sites directly influenced the dispersion of eggs by females . There was a significant difference (p <0.05) between the densities 2 * 4, 2 * 8, 2 * 16 and 4 * 16 both in laboratory and semi - field conditions (fig . 5). One female dispersed the eggs among 11 breeding sites, the highest number observed in the study . Skip oviposition behaviour, although widely used, may not occur, as observed in eight (7.4%) females . 5:mean and standard deviation of breeding sites colonised on the basis of available ones . Different letters mean statistical difference (kruskal - wallis p <0.05). One - by counting the number of eggs at each breeding site, we noticed that one of the sites usually received most of the eggs (40% or more of the total deposited eggs). Although all the breeding sites were identical and in semi - field and were not statistically different from each other, it one site generally received more eggs than the others . The amount of eggs that were deposited at the favourite site was similar in both laboratory and semi - field conditions . The average percentage of eggs that were deposited in the favourite site was significantly higher with a density of two breeding sites and did not vary among the other densities (fig . 6), showing that females tend to aggregate more eggs when there are only two available breeding sites . This number can be reduced by an increase in the dispersion among several breeding sites . There was a negative relationship between the number of colonised breeding sites and the percentages of eggs that were laid at the favourite breeding site (simple linear regression y = -0.0682x + 0.9256, f1,106 = 65.9, r = 0.38, p <0.001) (fig . 7). However, even those females that used many breeding sites seemed to deposit at least 40% of their eggs at the favourite breeding site, as shown by the dashed line (fig . 7). Nevertheless, comparing the percentages of eggs that were laid by the ae . Aegypti females of the two groups (above and below the regression lines, fig . 8), a significant difference was observed in both semi - field (t = -6.62, p <0.001) and laboratory (t = 2.87, p = 0.001) (fig . 7:percentage of eggs laid on favourite breeding site in relation to the number of colonised breeding sites under laboratory and semi - field conditions . 8:percentage of eggs laid in the favourite breeding site in laboratory by aedes aegypti female s who had higher frequency of breeding site colonisation (a) and smaller frequency (b). Percentage of eggs laid in the favourite breeding site in semi - field by ae . Aegypti female s who had higher frequency of breeding site colonisation (a) and smaller frequency (b). In other words, both environments (laboratory and semi - field) showed higher percentages of eggs laid at favourite breeding sites by females those that distributed their eggs in small numbers of containers . Although all the breeding sites were identical and in semi - field they were not statistically different from each other, one site generally received more eggs than the others . The results that were obtained under laboratory and semi - field conditions were similar, showing that the general aspects of the oviposition behaviour were maintained despite the individual variations that were observed between the tested females (wong et al . The variation in the number of eggs per female is common in experiments that assess a single gravidae . These variations are probably due to the blood - feeding efficiency (xue et al . 2008) and the size of the adults, which may interfere with the reproductive capacity of males and females (blackmore & lord 2000, ponlawat & harrington 2009). The average number of eggs was similar to that found by other authors (christophers 1960, madeira et al . As expected, the average number of eggs that were laid did not significantly differ between densities (2, 4, 8 and 16 breeding sites) or between environments (laboratory or semi - field). This result is probably due to the duration of the tests, which were performed between the third - seventh day after the consumption of blood meal, during which the females laid almost all of the eggs that were produced (gomes 2006, reiter 2007, chadee 2010). It was demonstrated for the first time that the behavioural response of females depended on the resources (number of breeding sites) that were provided to them . In an experiment with eight available containers, chadee (2010) noted that 87% of females used one to four breeding sites, with a maximum of seven . In our study, the number of containers that were used by the females increased following the increased availability of breeding sites under both laboratory and semi - field conditions . However, the number of breeding sites that were colonised seemed to stabilise at around five, even when there were 16 breeding sites available and reached a maximum of 11 under semi - field conditions . This result has epidemiological importance, as the search for breeding sites seems to be a crucial factor in the dispersal of the females and hence the diseases that they transmit (edman et al . The determination of the average and maximum number of breeding sites that were used by each female can aid in the development of methodologies for monitoring and controlling the vector . Therefore, further studies should be conducted to verify this behaviour in other environments and with different densities of breeding sites, given that few females colonised more than eight ovitraps . The results of the present study support the existence of the skip oviposition aegypti females, as previously observed (christophers 1960, fay & perry 1965, corbet & chadee 1993, apostol et al . 2003, reiter 2007, chadee 2010). The ability of ae . Aegypti females to distinguish potential breeding sites that will sustain the survival of their offspring during their development is a crucial factor in the life cycle of mosquitoes (zahiri & rau 1998). The selective pressure in favour of the females that make choices that may maximise the survival of their offspring (reiter 2007, harrington et al . Nevertheless, this behaviour does not occur sometimes, as observed in a few females in this study (7.4% of females) and by other authors (harrington & edman 2001, chadee 2010). The large proportion of eggs that were laid on water contradicts the findings of most authors, who reported that the number of eggs that were deposited on water is negligible compared to that deposited on the walls, filter paper or paddles of the breeding site (chadee & corbet 1987,chadee et al . However, a study in brazil demonstrated a large number of eggs that were deposited on water by females of two populations and at different humidities . The obtained figures were 42.9% and 57.3% (80% rh) and 61.4% and 63.2% (51% rh) for populations l and b, respectively (madeira et al . 2002). These findings were similar to the observations of the present work and almost 10 times higher than those reported by other authors (chadee & cobert 1987, chadee et al . Aegypti because females of the same population distributed their eggs in different available proportions in oviposition substrates, as observed in our study . Considering the behavioural plasticity of ae . Aegypti, it is possible that the oviposition in both environments (water or paddle / side walls of the breeding site) can be advantageous depending on the circumstance . In anthropic environments, females can lay eggs in a large range of ephemeral containers that are very susceptible to disturbance (reiter 2007). This behaviour can make the choice of laying most of their eggs out of water relevant in places that can only be achieved by increasing the level of the water and can offer more chances of the larvae hatching and reaching adulthood . Furthermore, deposition on the walls of the breeding sites may be an example of germ banking for the future of the ae . Aegyptipopulation (tsunoda et al . 2010). In unfavourable conditions, the maintenance of germ banking until the return of favourable periods may be more advantageous to the offspring than the immediate eclosion of the larvae . On the other hand, the behaviour of laying eggs directly on the water is also relevant to the vector from an epidemiological perspective, as it helps to maintain their populations when there is no rainwater to supply breeding sites, thus allowing the existence of floating populations of mosquitoes during the dry season (gomes et al . The deposition of eggs on water can also prevent predation by ants and cockroaches and the reduction of viability due to delays in hatching (madeira et al . This result could perhaps explain why the number of eggs on the water was higher in semi - field conditions, where the eggs would be more exposed to these attacks . The increase in the number of eggs on water as observed in this study could also be explained by the abundance of resources (breeding sites). The quick hatching of the larvae would increase the chances of the offspring finding the same available resources . However, to be considered a viable strategy, the eggs that were laid on the water surface must have hatching rates similar to those that were laid outside of the water . The literature is controversial on this issue because some authors have reported an extremely low hatching rate (2%) (silva et al . 2003), while others have found higher rates, such as from 47 - 53% (madeira et al . 2002) and from 73 - 80% (rey & oconnell 2014). Therefore, we believe that the deposition of eggs on the water may be, at least in certain cases, a good choice for the survival of ae . Aegypti . Our results are also relevant in the context of the strategies of monitoring vector populations . In brazil, ovitraps can be used to obtain the indices of infestation (ovitrap positivity index = number of ovitraps positive 100/number of ovitraps inspected and egg density index = number of eggs / number of positive ovitraps) by counting the number of eggs on the paddle of the installed ovitraps (gomes 1998, ms / svs 2009). In these surveys, the liquids (water or infusion grass) that are present in the ovitraps are discarded without performing any type of analysis . However, madeira et al . (2002) and the present study, both conducted in brazil, found high amounts of eggs in the ovitrap water . Additional studies should be performed to determine if this trend is maintained in other populations of the country and under field conditions . The present work demonstrated for the first time the existence of a favourite breeding site and showed that egg distribution was not equal . The term favourite breeding site was used to denote the breeding site that received the highest percentage of eggs . This site was observed in semi - field and in laboratory conditions and with different amounts of available breeding sites . The ae . Aegypti females usually choose the most productive containers to deposit most of their eggs . These containers are usually large, dark and unmanaged (maciel - de - freitas & loureno - de - oliveira 2011, wong et al . 2012). In the present study, this behaviour was maintained even when all of the breeding sites were identical . Either the females were able to recognise clues that were insignificant to us or this was such an intrinsic behaviour that it was maintained even when there was no advantage among the breeding sites . It is not known if the females lay their eggs in a single visit to the breeding site or if the breeding site receives a portion of the eggs and then the female returns to the same place and deposits eggs again . The observations on the proportion of eggs demonstrated the existence of two distinct patterns: (i) females lay many eggs at one favourite breeding site (40% or more) and spread the remainder at other breeding sites and (ii) females lay few eggs (less than 40%) at the favourite breeding site and display a greater potential for spreading the eggs remaining over other breeding sites . (2012), who observed an increase in the distribution of eggs in semi - field conditions when the highly productive containers were removed . The first pattern was more frequently observed in our study, although these two strategies can be important, depending on the condition of the breeding site and the environment in which the mosquitoes live . The behaviour of females laying most of their eggs at the favourite breeding site may favour the species in periods during which temporary breeding sites are scarce . This behaviour can also be advantageous when a female finds high - productivity breeding sites containing ideal conditions, such as large volumes and diameters, dark colouring and the presence of co - specific larvae . This behaviour would indicate the capacity to support large quantities of immature forms (harrington et al . 2008, maciel - de - freitas & loureno - de - oliveira 2011, wong et al . Females that choose other strategies, spreading the higher proportion of their eggs and depositing some at the favourite breeding site or spreading their eggs without considering the favourite breeding site, can favour their offspring during the rainy season, when there is a wide variety of breeding sites that are constantly supplied by rain water . Thus, the plasticity of behaviour that was observed in this study and in previous studies (madeira et al . The choice of a favourite breeding site and the deposition of large percentage of eggs in water were remarkable behaviours in the assessed population . Aegypti (madeira et al . 2002, paduan et al . 2006, hiragi et al . 2009) because they inhabit a variety of environmental conditions and present short life cycles . Aegyptimosquitoes contain populations of individuals of various origins that have experienced different selective pressures . Mosquito populations show great adaptability, primarily because their larval stages develop in different seasons and under different environmental conditions (becker 1989) and because different selective pressures of the environment can lead to populations presenting distinct characteristics and great genetic and behavioural plasticity (begon et al . 2011). To determine whether the oviposition behaviour is the result of phenotypic plasticity or intraspecific differences between different populations, studies are required . However, regardless of the origin of behavioural plasticity in the oviposition ofae . . Therefore, studies that elucidate the vector behaviour are important to the definition of control measures and despite efforts, little is known about the oviposition behaviour . Thereby, it is extremely important that further studies investigate these aspects, especially under field and different conditions . Finally, it is also important to understand the behaviour of mosquitoes that are used in the most modern and promising control techniques, such as those infected with the bacteria wolbachia and genetically modified mosquitoes.
The increase in the aged population, a westernized lifestyle, the development of diagnostic tools, and national surveillance programs for early prostate cancer detection may all have contributed to the increased detection rate of prostate cancer . Checking the serum prostate - specific antigen (psa) level and a digital rectal examination (dre) are the gold standards for prostate cancer screening . If repeated psa levels exceed 4 ng / ml or prostate nodule or asymmetry are found by dre, transrectal ultrasonography (trus)-guided needle biopsy is performed to pathologically confirm the diagnosis . A higher psa value is more frequently associated either with prostate cancer or with advanced disease, even though the opposite opinions have been reported in some cases (e.g., prostate cancer prediction with low psa in the case of elderly patients with abnormal dre and trus findings). More than 50% of men with a psa value higher than 10 ng / ml have extra - prostatic disease . Twenty percent of men with a psa higher than 20 ng / ml and 75% of those with a psa higher than 50 ng / ml are found to have pelvic lymph node involvement . Coagulopathy and immunosuppression can be problematic, and severe conditions are contraindications to prostate biopsy . After trus - guided needle biopsy, most infectious complications are limited to symptomatic urinary tract infection and low - grade febrile illness; however, recent studies showed that 1.4% of patients will go on to develop a febrile urinary tract infection, bacteremia, or acute prostatitis and require hospitalization for treatment with intravenous antibiotics . Bleeding is the most common complication seen after prostate biopsy, even with normal coagulation parameters . Other complications include acute urinary retention (aur), which may require temporary catheterization, the chance of which is increased with a prior history of benign prostatic hyperplasia and older age . Therefore, particularly in patients with extremely elevated serum psa levels and old age, prostate biopsy to confirm the diagnosis of prostate cancer should be carefully considered owing to possibly fatal complications . To prove the necessity for prostate biopsy in this group, we reviewed the outcomes of trus - guided prostate needle biopsy in patients with elevated psa levels . We grouped the patients according to their psa levels and calculated the positive predictive value (ppv). We retrospectively reviewed the medical records of 1,150 patients who underwent trus - guided prostate needle biopsy for the evaluation of prostate cancer from january 2000 to december 2010 . Patients with a pre - biopsy psa level less than 4 ng / ml were excluded from the study . We also excluded patients with acute prostatitis and acute urinary retention . In total, 1,121 patients were reviewed in this study . All patients took antibiotic prophylaxis (either po quinolones or third - generation cephalosporins) for 7 days, starting from the day before the biopsy . If any signs or symptoms suggesting urinary tract infection occurred, the procedure was canceled . Patients taking anticoagulation agents stopped the medication for at least 5 days before the biopsy . For most of the patients, 12-core or double sextant biopsy (i.e., right peripheral 3 core, right medial 3 core, left peripheral 3 core, and left medial 3 core) was performed . One or more cores were added if suspicious hypoechoic lesions or palpable nodules were present on ultrasound or dre . Some patients in poor general condition underwent standard sextant biopsy or 8-core biopsy . According to the pre - biopsy psa level, patients were divided into 6 groups (group a, 4 to 20 ng / ml; b, 20 to 40 ng / ml; c, 40 to 60 ng / ml; d, 60 to 80 ng / ml; e, 80 to 100 ng / ml; and f, above 100 ng / ml). The patients' age, pre - biopsy psa level, the result of biopsy (i.e., cancer cell type and the gleason score), and clinical or pathologic tnm stage were compared . The mean age of the patients with reported prostate cancer at biopsy was 65.8 years (range, 34 to 88 years) and that of the patients with a negative biopsy result was 70.1 years (range, 38 to 91 years). The mean psa level of the patients with a positive biopsy result was 16.12 ng / ml (range, 4.82 to 7,176 ng / ml). The mean psa level of patients with a negative result was 10.39 ng / ml (range, 4.12 to 85.5 ng / ml). The detection rate was gradually increased as the pre - biopsy psa level increased . For example, only 21.8% of group a (i.e., psa 4 to 20 ng / ml) had prostate cancer, whereas all 65 patients of group f (psa> 100 ng / ml) had cancer (positive predictive value=100.0%), as presented in fig . 1 . All 356 patients had prostate adenocarcinoma . Among them, 107 had high - grade cancer (gleason score 8). The percentage of high - grade cancer was 30% in total, which increased to 71% in group f (fig . 2). Magnetic resonance imaging (mri) was used to evaluate the local extent of disease and the possibility of lymph node involvement, and radionuclide bone scans were also performed to detect skeletal metastasis . If prostatectomy was performed, the radiologic findings and pathologic results were used to determine the tnm stage . Staging was possible for 311 of the 356 patients, whereas 45 patients had no record due to follow - up loss . A total of 156 patients (50%) had organ - confined cancer (table 1). Of the 56 patients who underwent workup for tnm stage in group f (psa> 100 ng / ml), 38 patients had either extra - prostatic diseases (ct3 - 4) or lymph node metastasis (n1), and 43 patients had positive bone scan metastasis (m1). Combining the results in group f, all patients had advanced prostate cancer (i.e., 4 patients [7%] had stage iii and the remaining 52 patients [93%] had stage iv cancer). No patient who underwent workup for tnm staging in group f had organ - confined cancer . An elevated serum psa level is one of the most important features suggestive of prostate cancer . In most cases, the psa cutoff of 4 ng / ml is a threshold for performing prostate biopsy . However, the test is painful and invasive, and various complications have been reported in the literature, albeit not common . Most of the complications were minor and did not require hospitalization, but serious life - threatening complications, such as sepsis, also occurred [10 - 13]. In general, the risk of prostate cancer is directly related to the psa level . Our analysis demonstrated that a serum psa level higher than 100 ng / ml was 100.0% accurate in predicting the presence of prostate cancer on tissue biopsy . Reported similar results in a large unscreened population . Using a subset of 716 patients with a psa value higher than 4 ng / ml, they found a positive predictive value of 98% for psa over 60 ng / ml . Gerstenbluth et al . Reported that, when increased to over 50 ng / ml, the serum psa level was 98.5% accurate in predicting cancer . Thus, they suggested that some highly selected patients may not need to undergo prostate biopsy before androgen deprivation therapy . There may be a disparity between these reports and ours, possibly resulting from racial differences . In our results, the ppv for psa over 50 ng / ml was merely 81.2%, which was lower than the value in the other western studies . In other words, the negative predictive value for psa over 50 ng / ml was higher . Also, the number of patients who had psa over 50 ng / ml was comparatively smaller, resulting in a difference in ppv from other western studies . In the study by egawa et al ., prostate cancer was present in 59.5% of patients with a psa of 10 ng / ml or higher . Shim et al . Also reported that the risk of positive biopsy for psa levels more than 100 ng / ml was 100%, which was quite similar to our results . Studies of korean populations have also documented a positive predictive value of 93.8% with a psa higher than 100 ng / ml and of 94.7% with a psa higher than 50 ng / ml, which increased to 100.0% in conjunction with an abnormal dre . In the present study, the detection rate was 100.0% in the subgroup of patients with a psa of 100 ng / ml or higher . In group e (psa, 80 to 100 ng / ml), one patient with a negative biopsy result was followed up for 6 months and a second prostate biopsy was performed, which confirmed prostate cancer . We did not include this patient in group f but rather in group e. this is because this study was conducted to verify the necessity for prostate biopsy in diagnosing prostate cancer; thus, we considered the result of the initial biopsy, not the repeated . Thus, patients with carcinoma reported at their repeated biopsy and not at their initial biopsy were classified as having a negative prostate cancer result . Gerstenbluth et al . Have suggested that a negative biopsy in patients with a psa value of higher than 20 ng / ml is often a false - negative . The likelihood of cancer is significantly increased if the psa level is persistently high at a subsequent measurement . The most important role of prostate biopsy is to confirm the diagnosis pathologically, but it also helps to obtain the histologic subtype and to estimate the extent of disease (e.g., the number of positive cores and percentage of positive cores). Therefore, if cancer is expected to be present in almost 100% of a specific patient group, then the value of biopsy is reduced merely to histologic grading and estimation of disease extent . In our review, all 65 patients in group f (psa> 100 ng / ml) had adenocarcinoma . Ninety percent of the patients had a gleason grade of 4 or higher (i.e., gleason score of 7 or higher), and radiologic studies showed that all the cancers were stage iii or iv (table 1). In addition, the characteristics of the patients in group f are presented in table 2 . The patient's age, body mass index, gleason score, and percentage of positive cores was investigated, but the results showed all patients to have high stages . Therefore, the information from the prostate biopsy (e.g., gleason score and the number of positive cores) does not affect the treatment strategy . If advanced disease is suspected by a super - high psa level and the results of imaging studies, definitive surgical treatment will not be an option . In our study, thus, if cancer is suspected in 100% of the patient group, and both disease extent and histologic subtype can be expected to be in a narrow range, undergoing a needle biopsy would be of little value for this specific group (i.e., patients with extremely high levels of serum psa). Inflammation due to infection has been found to produce significant psa elevation, and up to 6 to 8 weeks might be needed for the psa value to return to baseline . Psa elevations can also occur as the result of urethral procedures with instrumentation, or following prostatic manipulation, such as the dre . Therefore, in a patient with an increased psa level and any recent history of either acute infection, urinary retention, or urinary tract manipulation, prostate biopsy is not mandatory, and a period of time is required to obtain an accurate psa level [19 - 21]. The limits of our study are the relatively small number of patients and the retrospective design . To validate this cutoff value, a prospective study is needed to compare patients who underwent prostate biopsy with those who did not . Our results suggest the possibility for a biopsy - free diagnosis of prostate cancer, with the criterion of a psa level> 100 ng / ml and evidence of advanced disease in imaging studies . We still recommend that any patient who is considered a candidate for surgery or radiation, despite a high psa level, should undergo biopsy . Nevertheless, because prostate needle biopsy can be associated with several adverse effects, if elderly patients with poor general medical condition present with an extremely elevated serum psa level, a tissue diagnosis of prostate cancer may not be required for starting androgen deprivation therapy.
In aging males, benign prostatic hyperplasia (bph) is one of the most common and bothersome diseases influencing quality of life . For decades, transurethral resection of the prostate (turp) has been recognized as the standard treatment for bph . In recent years, laser treatment of bph has challenged turp as the result of advances in laser technology, better understanding of tissue - laser interactions, and growing clinical experience . Although green light laser photoselective vaporization has been proved to be a safe and effective surgical procedure comparable to turp, photoselective vaporization has the limitations of providing no tissue specimen for histological evaluation and having a significantly slower speed of tissue ablation . According to the results of recent randomized studies, holmium laser enucleation of the prostate (holep) can be applied to all cases of bph regardless of prostate size and has been proved to be an alternative to turp or open prostatectomy . The most beneficial aspect of holep may be an enucleation function similar to that of open prostatectomy . The recently introduced thulium laser has a wavelength similar to that of the holmium laser but has been found to be superior in spatial beam quality, precise incision, and wave continuity . In comparative studies, thulium laser enucleation of the prostate (thulep) showed results comparable to those of holep . In this study, we introduce a new one - lobe enucleation technique of thulep . We predicted that the technique might even better reproduce the enucleation ability of open prostatectomy . From june 2013 to may 2014, a total of 47 symptomatic bph patients who underwent the all - in - one technique of thulep were evaluated . Inclusion criteria were as follows: maximal urine flow rate (qmax) less than 15 ml / s or postvoid residual urine volume (pvr) above 50 ml or repeated urinary retention or lower urinary tract symptoms with an international prostate symptom score (ipss) above 7 . Patients with prostate cancer, urethral stricture, or a previous history of urologic surgery were excluded . All patients were assessed with medical history, digital rectal examination (dre), ipss, transrectal ultrasonography (trus), serum prostate - specific antigen (psa), qmax, and pvr before surgery and 1 month after surgery . Perioperative data (i.e., operative time, enucleation time, morcellation time, resected tissue weight, serum hemoglobin decrease, catheterization day, postoperative complications) were recorded . To evaluate enucleation efficacy, we assessed the psa reduction ratio ([preoperative psa level - postoperative psa level]/[preoperative psa level]), transitional zone volume reduction ratio (resected tissue weight / preoperative transitional zone volume), and enucleation failure rate . Enucleation failure was defined as cases in which postoperative trus revealed remnant adenoma in the transitional zone . In patients who had risk factors for prostate cancer (e.g., psa value above 4 ng / ml, nodule on dre, or hypoechoic lesion on trus) patients who proved to have prostate cancer through preoperative biopsy or postoperative biopsy were also excluded . The significance level was defined as a p - value <0.05 . The instruments used were a 70-w continuous - wave tm: yttrium aluminium garnet laser (revolix, lisa laser products ohg, katlenburg - lindau, germany) and a 26-french continuous flow resectoscope (richard wolf medical instruments, vernon hills, il, usa). The enucleated prostatic tissues were morcellated by use of a piranha morcellator (richard wolf medical instruments, vernon hills, il, usa). The surgical steps of the all - in - one technique of thulep are follows . After inspection of the urethral sphincter, a marking incision is made at 12 o'clock at the apex to ensure the safety of the sphincter (fig . Then the superior 12 o'clock incision line and the end of the inverted - u - shaped incision line at inferior 5 and 7 o'clock are connected circumferentially (fig . After confirming the safety of the urethral sphincter once more, a deep incision is made along the lateral circumferential line until the surgical capsule is visible starting from 5 or 7 o'clock to 12 o'clock . If the circumferential surgical capsule is wholly identified, the enucleation process goes on to the bladder neck along the obtained circumferential surgical capsule with use of the thulium laser for the incision and coagulation and the beak of the resectoscope for blunt dissection . Near the bladder neck, freeing of the enucleated prostatic adenoma proceeds from 12 o'clock in a latero - inferior direction . The 5 to 7 o'clock area, the median lobe, is freed last (fig . After freeing of the entire prostatic lobe circumferentially from the surgical capsule, the last step before morcellation is to ensure that there is no significant bleeding . If there is no significant bleeding, the morcellator is then inserted into the bladder to morcellate the enucleated prostatic adenoma . From june 2013 to may 2014, a total of 47 symptomatic bph patients who underwent the all - in - one technique of thulep were evaluated . Inclusion criteria were as follows: maximal urine flow rate (qmax) less than 15 ml / s or postvoid residual urine volume (pvr) above 50 ml or repeated urinary retention or lower urinary tract symptoms with an international prostate symptom score (ipss) above 7 . Patients with prostate cancer, urethral stricture, or a previous history of urologic surgery were excluded . All patients were assessed with medical history, digital rectal examination (dre), ipss, transrectal ultrasonography (trus), serum prostate - specific antigen (psa), qmax, and pvr before surgery and 1 month after surgery . Perioperative data (i.e., operative time, enucleation time, morcellation time, resected tissue weight, serum hemoglobin decrease, catheterization day, postoperative complications) were recorded . To evaluate enucleation efficacy, we assessed the psa reduction ratio ([preoperative psa level - postoperative psa level]/[preoperative psa level]), transitional zone volume reduction ratio (resected tissue weight / preoperative transitional zone volume), and enucleation failure rate . Enucleation failure was defined as cases in which postoperative trus revealed remnant adenoma in the transitional zone . In patients who had risk factors for prostate cancer (e.g., psa value above 4 ng / ml, nodule on dre, or hypoechoic lesion on trus) patients who proved to have prostate cancer through preoperative biopsy or postoperative biopsy were also excluded . The instruments used were a 70-w continuous - wave tm: yttrium aluminium garnet laser (revolix, lisa laser products ohg, katlenburg - lindau, germany) and a 26-french continuous flow resectoscope (richard wolf medical instruments, vernon hills, il, usa). The enucleated prostatic tissues were morcellated by use of a piranha morcellator (richard wolf medical instruments, vernon hills, il, usa). The surgical steps of the all - in - one technique of thulep are follows . After inspection of the urethral sphincter, a marking incision is made at 12 o'clock at the apex to ensure the safety of the sphincter (fig . Then the superior 12 o'clock incision line and the end of the inverted - u - shaped incision line at inferior 5 and 7 o'clock are connected circumferentially (fig . After confirming the safety of the urethral sphincter once more, a deep incision is made along the lateral circumferential line until the surgical capsule is visible starting from 5 or 7 o'clock to 12 o'clock . If the circumferential surgical capsule is wholly identified, the enucleation process goes on to the bladder neck along the obtained circumferential surgical capsule with use of the thulium laser for the incision and coagulation and the beak of the resectoscope for blunt dissection . Near the bladder neck, freeing of the enucleated prostatic adenoma proceeds from 12 o'clock in a latero - inferior direction . The 5 to 7 o'clock area, the median lobe, is freed last (fig . After freeing of the entire prostatic lobe circumferentially from the surgical capsule, the last step before morcellation is to ensure that there is no significant bleeding . If there is no significant bleeding, the morcellator is then inserted into the bladder to morcellate the enucleated prostatic adenoma . After inspection of the urethral sphincter, a marking incision is made at 12 o'clock at the apex to ensure the safety of the sphincter (fig . Then the superior 12 o'clock incision line and the end of the inverted - u - shaped incision line at inferior 5 and 7 o'clock are connected circumferentially (fig . After confirming the safety of the urethral sphincter once more, a deep incision is made along the lateral circumferential line until the surgical capsule is visible starting from 5 or 7 o'clock to 12 o'clock . If the circumferential surgical capsule is wholly identified, the enucleation process goes on to the bladder neck along the obtained circumferential surgical capsule with use of the thulium laser for the incision and coagulation and the beak of the resectoscope for blunt dissection . Near the bladder neck, freeing of the enucleated prostatic adenoma proceeds from 12 o'clock in a latero - inferior direction . The 5 to 7 o'clock area, the median lobe, is freed last (fig . After freeing of the entire prostatic lobe circumferentially from the surgical capsule, the last step before morcellation is to ensure that there is no significant bleeding . If there is no significant bleeding, the morcellator is then inserted into the bladder to morcellate the enucleated prostatic adenoma . A total of 47 patients who underwent the all - in - one thulep technique were included in this study . Mean operative time was 82.133.3 minutes, mean enucleation time was 52.721.7 minutes, mean morcellation time was 8.27.0 minutes, mean resected tissue weight was 36.924.6 g, mean hemoglobin decrease was 0.40.8 g / dl, and mean number of catheterization days was 2.82.0 days (table 1). Preoperative mean trus prostate volume and mean transitional zone volume were 66.938.6 cm and 40.626.4 cm, respectively . Mean psa was 7.815.9 ng / ml, mean ipss was 24.87.9, mean qmax was 7.34.5 ml / s, and mean pvr volume was 137.6201.0 ml . After 1 month, mean trus prostate volume was 11.710.0 cm, mean transitional zone volume was 0.94.8 cm, mean psa was 0.50.4 ng / ml, mean ipss was 17.510.0, mean qmax was 15.67.8 ml / s, and mean pvr was 30.020.7 ml . After 3 months, mean ipss was 11.57.1, mean qmax was 15.89.0 ml / s, and mean pvr was 27.317.5 ml . All 1-month follow - up values were significantly improved over the corresponding preoperative values (p<0.05). The 3-month follow - up ipss was significantly improved compared with the 1-month follow - up score (p<0.05). The psa reduction ratio was 0.810.23 and the transitional zone volume reduction ratio was 0.920.34 . There were only 2 cases of enucleation failure; the enucleation failure rate was 4.3% (table 3). There were also no cases of capsular perforation, ureteral orifice injury, or urinary tract infection . Two cases of superficial bladder injury occurred while performing morcellation (4.3%), and there were 8 cases of temporary urinary retention (17.0%), 1 case of stress urinary incontinence (2.1%), and 1 case of urethral stricture . Prostate enucleation with a laser is a safe, effective, and minimally invasive technique regardless of prostate size, and recent studies have reported that it can substitute for turp or open prostatectomy . In particular, both thulium and holmium lasers have high efficacy and safety in relieving the lower urinary tract symptoms of bph patients . During the past 2 decades, holmium laser surgery for bph has gradually advanced in the fields of both laser technology and surgical techniques . In the 1990s, since the introduction of the tissue morcellator, holmium laser ablation and resection has largely been superseded by holep . Since then many studies have reported promising results that support the excellency of holep for bph surgery . To date, many surgeons have adapted the' three - lobe' technique for holep . To facilitate enucleation, krambeck et al . And baazeem et al . Similar to the holmium laser, the thulium laser works with a wavelength of 2013 nm, which can be easily absorbed into water, especially interstitial water . The waves of the thulium laser are more continuous than those of the holmium laser and therefore provide more effective hemostasis . In addition, it provides accurate resection with sufficient vaporization, and the moving laser probe maximizes vaporization but reduces the heat applied to tissue . The development of thulium laser prostate surgery has proceeded very similarly to that of holmium laser surgery . Four different techniques have been described so far: thulium vaporization of the prostate, thulium vaporesection (thuvarp), thulium vapoenucleation (thuvep), and thulium enucleation (thulep). The results showed that thuvarp had the same therapeutic effect as turp and had the advantages of a shorter hospital stay and shorter catheter indwelling time . However, thuvarp had a limitation in that the operation time took longer as the prostate size increased . Thus, thuvarp is more suitable for a relatively small prostate . To overcome the prolonged operation time in a large prostate, bach et al . Reported the technique of thuvep, and its functional outcomes were comparable with those of holep . Also, there was no limitation in prostate size . They said that thulep offers the opportunity for maximum removal of obstruction of the prostate urethra similar to open prostatectomy but with maximum control of hemostasis . As mentioned above, the surgical techniques of the holmium and thulium laser have been developed to reproduce and resemble the enucleation techniques of open prostatectomy . It is expected that greater tissue removal will result in better functional results with regard to improvement of qmax, pvr, and the re - treatment rate . Our all - in - one technique can mostly reproduce the enucleation techniques of open prostatectomy . We expect that this new technique may have three advantages over the usual two- or three - lobe technique: shortening the operation time, reducing blood loss, and improving the effectiveness of enucleation . However, these advantages were not proven in the present study because this was a preliminary study and contains our initial experience . Theoretically, however, we can reduce the incision and dissecting surface, and the possibility of encountering unexpected bleeding can be lowered because the 5 or 7 o'clock incision from the apex to the bladder neck of the ordinary technique is not needed in the all - in - one technique . In this study, we reported comparable results . The mean enucleation time was 52.7 minutes and the mean hemoglobin decrease was 0.4 g / dl . Also, there was no transfusion . To evaluate enucleation efficacy, we used three parameters: psa reduction ratio, transitional zone volume reduction ratio, and enucleation failure rate . The psa reduction ratio was 0.81, which was comparable to previously reported thulep or holep results . There are no other reports that used the transitional zone volume reduction ratio (0.92) and the enucleation failure rate (4.3%) as the parameters for assessing enucleation efficacy . Although more long - term data and well - designed comparative study are needed, our initial results of the all - in - one technique are promising . We believe that the all - in - one technique for thulep will be the most effective surgical management for bph . We introduce a novel one - lobe technique of thulep, the all - in - one technique, and report its initial results . Our initial results prove that the all - in - one technique is not inferior to another technique for symptomatic bph surgery and is a feasible and promising technique . Future well - designed comparative study and long - term data can prove that this new technique has some advantages in terms of operation time, hemostasis, and enucleation efficacy . An accompanying video can be found in the' urology in motion' section of the journal homepage (www.kjurology.org). The supplementary video clips can also be accessed by scanning a qr code located on the fig . 1 of this article, or be available on youtube (http://youtu.be/shj6glar2my).
All the solvents and chemicals were purchased from merck, nice chemicals and sd fine chemicals . Melting points were determined by using melting point apparatus mp - ds tid 2000 v and the values were uncorrected . Reactions were monitored by thin layer chromatography (tlc methanol: acetone, 6: 4) on pre - coated silica gel g plates using iodine vapor as visualizing agent . Ir spectra were recorded on jasco ft / ir-140 spectrophotometer by using kbr pellets technique . Pmr spectra were recorded using brucker ft - nmr-500 mhz spectrophotometer by using dmso as solvent and tms as internal standard . The experimental protocol for the pharmacological screening on rats were done with an institutional animal ethics committee, k.m . College of pharmacy, madurai, india (reg no: 661/02/c / cpcsea). 2-acetyl benzimidazole (0.01 mol) and appropriately substituted aromatic aldehydes (0.012 mol) were mixed in ethanol (20 ml) containing 10% aq . Koh (8 ml) and magnetically stirred the solution constantly at room temperature for 10 h. the whole mixture was transferred into a 100 ml ice cold water and acidified with dil . The solid formed was washed, filtered and dried, recrystallized from absolute ethanol . To a solution of (4a f) (0.01 mol) was suspended in 7 ml acetic acid . To this barbituric acid (0.01 mol) the reaction progress was monitored by tlc (methanol: acetone, 6: 4). The crude product was filtered, washed with water and recrystallized from methanol . Physical characterization of all the synthesized derivatives is shown in table 1 . Physical characterization and insilico screening of (5a - f) 3230(nhstr),1693(c = o),1587 (c = n). Hnmr(dmso - d6), ppm9.1(1h, s, nhbenzimidazole), 8.0 - 8.4(2h, s, pyrimidinenh)6.4 - 8.1(11h, m,9arh,2ch = ch).m.s: m / z=359.21 (m + 1). Hnmr(dmso - d6), ppm:8.9(1h, s, nhbenzimidazole),8.4 - 8.6(2h, s, pyrimidinenh)6.4 - 8.3(11h, m,9arh,2ch = ch).m.s: m / z=394.1 (m + 2) 3239(nhstr),1681(c = o),1576(c = n), hnmr(dmso - d6), ppm:8.9(1h, s, nhbenzimidazole),8.4 - 8.6(2h, s, pyrimidinenh),6.4 - 8.3(10h, m,8arh,2ch = ch).3.3(3h, s, och3). M.s: hnmr(dmso - d6), ppm:8.9(1h, s, nhbenzimidazole),8.5 - 8.7(2h, s, pyrimidinen),6.3 - 8.4(10h, m,8arh,2ch = ch).3.3(6h, s, n(ch3)2 m.s: m / z=402.56 (m + 1) 3227(nhstr).1678(c = o),1569(c = n),1310(ar - no2). Hnmr(dmso - d6),ppm:8.5(1h, s, nhbenzimidazole),8.2 - 8.4(2h, s, pyrimidinenh),6.4 - 8.4(10h, m,8arh,2ch = ch) m.s: m / z=405.56 (m + 2) 3379(aroh),3231(nhstr),1680(c = o),1562(c hnmr(dmso - d6),ppm:9.1(1h, s, nhbenzimidazole),8.4 - 8.8(2h, s, pyrimidinenh)6.4 - 8.5(10h, m,8arh,2ch = ch),5.62(1h, s, aro - h) m.s: m / z=375.65 (m + 1) estimation of general biological potential for drug - like compounds on the basis of their structural formulae can be performed with a computer program pass (prediction of activity spectra for substances) that predicts more than 780 pharmacological effects . All the structures of the derivatives were built by acd chemsketch version 12.0 and saved as mol format . These can mol formats can be imported into the pass for prediction of the biological activity . This program was based on a robust analysis of structure - activity relationships in a heterogeneous training set . A biological spectrum for a substance is a list of biological activity types for which the probability to be revealed (pa) and not to be revealed (pi) values are independent and their values ranges from 0 to 1 . The more is the pa value, the less is the probability of false positives in the set of compounds selected for study. [1315] all the titled derivatives predicted a biological activity of mucomembranous protector nature with a pa values more than 0.70 . The study was carried out for four days after administration of the treated dose half hour after the rats were treated with aspirin 200 mg / kg . This process was carried out for three days . On the third day after administration of drug, the rats were sacrificed four hours later by cervical dislocation and the esophagi were clamped, the stomach was exposed carefully, opened along the greater curvature, the luminal contents were removed, and the total volume of gastric secretion, total acidity, free acidity were estimated by titration method . The percentage of ulcer inhibition was determined as follows: values are expressed as mean sem, values were found by using one - way anova followed by newman keul's multiple range tests [table 2]. 2-acetyl benzimidazole (0.01 mol) and appropriately substituted aromatic aldehydes (0.012 mol) were mixed in ethanol (20 ml) containing 10% aq . Koh (8 ml) and magnetically stirred the solution constantly at room temperature for 10 h. the whole mixture was transferred into a 100 ml ice cold water and acidified with dil . To a solution of (4a f) (0.01 mol) was suspended in 7 ml acetic acid . To this barbituric acid (0.01 mol) the reaction progress was monitored by tlc (methanol: acetone, 6: 4). Physical characterization and insilico screening of (5a - f) 3230(nhstr),1693(c = o),1587 (c = n). Hnmr(dmso - d6), ppm9.1(1h, s, nhbenzimidazole), 8.0 - 8.4(2h, s, pyrimidinenh)6.4 - 8.1(11h, m,9arh,2ch = ch).m.s: m / z=359.21 (m + 1). Hnmr(dmso - d6), ppm:8.9(1h, s, nhbenzimidazole),8.4 - 8.6(2h, s, pyrimidinenh)6.4 - 8.3(11h, m,9arh,2ch = ch).m.s: m / z=394.1 (m + 2) 3239(nhstr),1681(c = o),1576(c = n), hnmr(dmso - d6), ppm:8.9(1h, s, nhbenzimidazole),8.4 - 8.6(2h, s, pyrimidinenh),6.4 - 8.3(10h, m,8arh,2ch = ch).3.3(3h, s, och3). Hnmr(dmso - d6), ppm:8.9(1h, s, nhbenzimidazole),8.5 - 8.7(2h, s, pyrimidinen),6.3 - 8.4(10h, m,8arh,2ch = ch).3.3(6h, s, n(ch3)2 m.s: m / z=402.56 (m + 1) 3227(nhstr).1678(c = o),1569(c = n),1310(ar - no2). Hnmr(dmso - d6),ppm:8.5(1h, s, nhbenzimidazole),8.2 - 8.4(2h, s, pyrimidinenh),6.4 - 8.4(10h, m,8arh,2ch = ch) m.s: m / z=405.56 (m + 2) 3379(aroh),3231(nhstr),1680(c = o),1562(c hnmr(dmso - d6),ppm:9.1(1h, s, nhbenzimidazole),8.4 - 8.8(2h, s, pyrimidinenh)6.4 - 8.5(10h, m,8arh,2ch = ch),5.62(1h, s, aro - h) m.s: m / z=375.65 (m + 1) hnmr(dmso - d6), ppm9.1(1h, s, nhbenzimidazole), 8.0 - 8.4(2h, s, pyrimidinenh)6.4 - 8.1(11h, m,9arh,2ch = ch).m.s: m / z=359.21 (m + 1). Hnmr(dmso - d6), ppm:8.9(1h, s, nhbenzimidazole),8.4 - 8.6(2h, s, pyrimidinenh)6.4 - 8.3(11h, m,9arh,2ch = ch).m.s: m / z=394.1 (m + 2) 3239(nhstr),1681(c = o),1576(c = n), hnmr(dmso - d6), ppm:8.9(1h, s, nhbenzimidazole),8.4 - 8.6(2h, s, pyrimidinenh),6.4 - 8.3(10h, m,8arh,2ch = ch).3.3(3h, s, och3). Hnmr(dmso - d6), ppm:8.9(1h, s, nhbenzimidazole),8.5 - 8.7(2h, s, pyrimidinen),6.3 - 8.4(10h, m,8arh,2ch = ch).3.3(6h, s, n(ch3)2 m.s: m / z=402.56 (m + 1) hnmr(dmso - d6),ppm:8.5(1h, s, nhbenzimidazole),8.2 - 8.4(2h, s, pyrimidinenh),6.4 - 8.4(10h, m,8arh,2ch = ch) m.s: m / z=405.56 (m + 2) hnmr(dmso - d6),ppm:9.1(1h, s, nhbenzimidazole),8.4 - 8.8(2h, s, pyrimidinenh)6.4 - 8.5(10h, m,8arh,2ch = ch),5.62(1h, s, aro - h) m.s: m / z=375.65 (m + 1) estimation of general biological potential for drug - like compounds on the basis of their structural formulae can be performed with a computer program pass (prediction of activity spectra for substances) that predicts more than 780 pharmacological effects . All the structures of the derivatives were built by acd chemsketch version 12.0 and saved as mol format . These can mol formats can be imported into the pass for prediction of the biological activity . This program was based on a robust analysis of structure - activity relationships in a heterogeneous training set . A biological spectrum for a substance is a list of biological activity types for which the probability to be revealed (pa) and not to be revealed (pi) values are independent and their values ranges from 0 to 1 . The more is the pa value, the less is the probability of false positives in the set of compounds selected for study. [1315] all the titled derivatives predicted a biological activity of mucomembranous protector nature with a pa values more than 0.70 . The study was carried out for four days after administration of the treated dose half hour after the rats were treated with aspirin 200 mg / kg . This process was carried out for three days . On the third day after administration of drug, the rats were sacrificed four hours later by cervical dislocation and the esophagi were clamped, the stomach was exposed carefully, opened along the greater curvature, the luminal contents were removed, and the total volume of gastric secretion, total acidity, free acidity were estimated by titration method . The ulcer index was calculated according to the method of ganguly and bhatnagar . The percentage of ulcer inhibition was determined as follows: values are expressed as mean sem, values were found by using one - way anova followed by newman keul's multiple range tests [table 2]. The present study described a knoevenagel condensation between a benzimidazole chalcone and barbituric acid by using experimental protocol as shown in figure 1 . The structures of all the final derivatives were established on the basis of spectral analysis . Ir spectrum of the 5-[(2e)-1-(1h - benzimidazol-2-yl)-3-phenylprop-2-en-1-ylidene] pyrimidine-2,4,6 (1h, 3h, 5h)-trione 5b showed a strong absorption band at 3232 cm corresponding to benzimidazole nh and absorption at 2910 and 2878 cm corresponding to pyrimidine nh / nh . A sharp absorption at 1678 cm corresponds to carbonyl stretching and 771 cm due to aryl chloride . The hnmr spectra showed the singlet peak at 8.4, and 8.6 was assigned to pyrimidine nh / nh . The singlet peak was at 8.9 corresponding to benzimidazole nh . The doublet for vicinal protons has been seen along with the aromatic multiplet between 6.4 and 8.3 ppm . Mass spectrum of compound 5b revealed the molecular ion peak [m+2] at m / z 394 corresponding to the molecular mass of the compound . Mass fragmentation pattern of 5b gave a base peak of m / z 58 which corresponds to the molecular formula of ch2n2o . This splitting of the urea fragment from the 5b also made full agreement with the structure of final barbitone moiety . Scheme of synthetic route of the titled compounds the pass program predicted the mucomembranous protector nature probabilities 5-[(2e)-1-(1h - benzimidazol-2-yl)-3-substituted phenylprop-2-en-1-ylidene] pyrimidine-2, 4, 6 (1h, 3h, 5h)-triones (5a f) showing the following rank order 5d> 5b> 5c> 5a> 5e> 5f . It is important to note that compounds 5d, 5b and 5c showed a percentage protection of (69.58, 69.56 and 67.17 at a dose of 50 mg / kg b.w .) When compared to standard omeprazole (77.37%, 2 mg / kg b.w . ). This suggested a good correlation between the predicted and observed activity score in the final candidates . Administration of 200 mg / kg asa suspension intragastrically consistently caused hemorrhagic lesions in the mucosa of the glandular stomach, indicating true ulcer formation as stated in histological findings . The stomach specimen of aspirin - treated rats was characterized by complete disruption of protective mucosal layer [figure 2]. The tissue of aspirin - treated rats had shown that some epithelial cells in the ulcer margin had proliferated and migrated over and into the ulcer crater, which was strongly infiltrated by inflammatory cells, fibroblasts and endothelial cells indicating complete disruption of gastric epithelial layer . Scanning of stomach specimens using electron microscope revealed that in the rats treated with omeprazole, 5b and 5d [figures 35], there was no injury observed in stomach mucosa, which is identical to that of the control animal [figure 6]. The present study described a knoevenagel condensation between a benzimidazole chalcone and barbituric acid by using experimental protocol as shown in figure 1 . The structures of all the final derivatives were established on the basis of spectral analysis . Ir spectrum of the 5-[(2e)-1-(1h - benzimidazol-2-yl)-3-phenylprop-2-en-1-ylidene] pyrimidine-2,4,6 (1h, 3h, 5h)-trione 5b showed a strong absorption band at 3232 cm corresponding to benzimidazole nh and absorption at 2910 and 2878 cm corresponding to pyrimidine nh / nh . A sharp absorption at 1678 cm corresponds to carbonyl stretching and 771 cm due to aryl chloride . The hnmr spectra showed the singlet peak at 8.4, and 8.6 was assigned to pyrimidine nh / nh . The singlet peak was at 8.9 corresponding to benzimidazole nh . The doublet for vicinal protons has been seen along with the aromatic multiplet between 6.4 and 8.3 ppm . Mass spectrum of compound 5b revealed the molecular ion peak [m+2] at m / z 394 corresponding to the molecular mass of the compound . Mass fragmentation pattern of 5b gave a base peak of m / z 58 which corresponds to the molecular formula of ch2n2o . This splitting of the urea fragment from the 5b also made full agreement with the structure of final barbitone moiety . The pass program predicted the mucomembranous protector nature probabilities 5-[(2e)-1-(1h - benzimidazol-2-yl)-3-substituted phenylprop-2-en-1-ylidene] pyrimidine-2, 4, 6 (1h, 3h, 5h)-triones (5a f) showing the following rank order 5d> 5b> 5c> 5a> 5e> 5f . It is important to note that compounds 5d, 5b and 5c showed a percentage protection of (69.58, 69.56 and 67.17 at a dose of 50 mg / kg b.w .) When compared to standard omeprazole (77.37%, 2 mg / kg b.w . ). This suggested a good correlation between the predicted and observed activity score in the final candidates administration of 200 mg / kg asa suspension intragastrically consistently caused hemorrhagic lesions in the mucosa of the glandular stomach, indicating true ulcer formation as stated in histological findings . The stomach specimen of aspirin - treated rats was characterized by complete disruption of protective mucosal layer [figure 2]. The tissue of aspirin - treated rats had shown that some epithelial cells in the ulcer margin had proliferated and migrated over and into the ulcer crater, which was strongly infiltrated by inflammatory cells, fibroblasts and endothelial cells indicating complete disruption of gastric epithelial layer . Scanning of stomach specimens using electron microscope revealed that in the rats treated with omeprazole, 5b and 5d [figures 35], there was no injury observed in stomach mucosa, which is identical to that of the control animal [figure 6]. In conclusion, a series of some novel 5-[(2e)-1-(1h - benzimidazol-2-yl)-3-substituted phenylprop-2-en-1-ylidene] pyrimidine-2, 4, 6 (1h, 3h, 5h)-triones were synthesized . We also demonstrated that most of the titled compounds showed moderate in vivo antiulcer activity but all the derivatives exhibited a good mucomembranous protector . The study has also shown an increase probability of compounds to be biologically active if they are selected on the basis of pass prediction.
Peripancreatic fluid collections (pfcs) can develop secondary to either fluid leakage or liquefaction of pancreatic necrosis following acute pancreatitis, chronic pancreatitis, surgery, or abdominal trauma.14 previously focusing on the original atlanta classification of acute pancreatitis,5 pfcs include acute fluid collections, acute and chronic pancreatic pseudocysts, pancreatic abscesses, and pancreatic necrosis . Pancreatic abscess to define a localized collection of purulent material without significant necrotic material . However, since this finding is extremely uncommon, the term pancreatic abscess was confusing even investigators of pancreatic diseases . In 2013, the revised atlanta classification proposed to clarify several issues from the original atlanta classification.6 the revised atlanta classification classified local complications mostly followed by acute pancreatitis into four types according to pathological conditions and timing as follows: 1) acute peripancreatic fluid collection (apfc); 2) acute necrotic collection (anc) (sterile or infected); 3) pancreatic pseudocyst (pp); and 4) walled - off necrosis (won) (sterile or infected),6 in this classification, the term pancreatic abscess was removed and divided into infected pps and wons based on their component and radiologic images.6 until 2013, an infected pp was lumped together with an infected won in the same category as a pancreatic abscess . Thus, an infected anc / pp or won must be set apart from apfc, sterile pp or won based on the revised atlanta classification6 because the strategy of treatment is markedly different (table 1). The outcome of endoscopic drainage was significantly worse for won compared with pp, with significantly fewer collection disappearances and more complications.7 even if the pp should not always be treated according to the american society for gastrointestinal endoscopy guideline,8 the indication for drainage of pp are symptoms (abdominal pain, early satiety), complications (infection, bleeding, rupture), obstruction of a surrounding hollow viscous (gastric, duodenal, or biliary obstruction), or enlarged pp . Drainage of pp was also recommended if the pps were larger than 6 cm, continued to increase in size or did not resolve after 4 to 6 weeks7 as well as symptomatic lesions . Infected anc was also recommended for drainage similarly to an infected pp . On the other hand, infected wons, which consisted of a mature, encapsulated collection of pancreatic and/or peripancreatic necrosis that has developed a well - defined inflammatory wall, were recommended for not only drainage but also necrosectomy if needed . At present, endoscopic drainages are popular as a minimally invasive alternative to surgical or percutaneous drainage for pfc management . Of the endoscopic drainages for pfcs, endoscopic ultrasound - guided transluminal drainage (eus - td) has become the standard and safe procedure in many centers for the nonsurgical treatment of pfcs because it can provide a safe puncture avoiding intervening blood vessels . Thus far, single or multiple plastic stents (combined with a nasocystic catheter) have commonly been used for the treatment of pfcs for eus - td . More recently, the use of covered self - expandable metallic stents (csemss) has provided a safer and more efficient approach route for internal drainage . In this review, we focus on the best approach and stent to use in endoscopic drainage for pfcs on the basis of the original atlanta classification5 because of lack of clinical results confirming the revised atlanta classification.6 a recent retrospective study regarding nonsurgical approaches percutaneous versus endoscopic transmural drainage (conventional direct transluminal drainage by forward - viewing endoscopy [ctd] or eus - td)to symptomatic pp revealed no significant difference between technical success rates in treating pp.9 however, percutaneous transmural drainage was associated with a higher reintervention rate, longer hospital stay, and increased number of follow - up abdominal imaging studies.9 therefore, endoscopic transmural drainage should be the preferred modality for the drainage of symptomatic pp compared with percutaneous drainage . A recent prospective randomized controlled trial regarding surgical drainage versus eus - td for symptomatic pp revealed no difference in treatment success, complications, or reinterventions between the surgical and eus - td groups, the length of hospital stay was shorter, the physical and mental health scores were better, and the total mean costs were lower for the eus - td group.10 because none of the patients randomized to eus - td developed pp recurrence at the follow - up evaluation, there was no evidence to suggest that surgical drainage is superior to eus - td for pp drainage . Thus, endoscopic drainage for pp drainage has become an effective alternative treatment to percutaneous and surgical drainage . Endoscopic drainage is now considered to be the first - line approach for treating symptomatic pp due to its less invasiveness, lower reinterventions, lower morbidity rate, and shorter hospital stay . However, we should consider that surgical treatment still has an important role in terms of adjunctive or salvage therapy if endoscopic or percutaneous intervention fails . Endoscopic drainage of pp consists of ctd, transpapillary drainage (tpd) and eus - td . In a web - based u.s . Survey that identified the american society for gastrointestinal endoscopy members who performed pp drainage in 2006, eus - td was used only by 56% of u.s . Endoscopists and 43% by international endoscopists.11 tpd requires that the pp communicate with the main pancreatic duct and that it has few septations to permit complete drainage . Pancreatic duct strictures or disruption, if identified, may be dilated, after which a single plastic stent is placed into the main pancreatic duct . It is also crucial to evaluate for the presence of a pancreatic fistula, which if present, should be initially treated by pancreatic duct stenting . If the pancreatic fistula does not resolve after a prolonged period of pancreatic duct stenting, endoscopic sealing with n - butyl-2-cyanoacrylate can be considered.12 a recent prospective cohort study of patients with refractory pancreatic duct strictures revealed that the use of a wire - guided diathermic dilator is feasible and safe . Wire - guided diathermic dilator treatment may be considered a new standard alternative procedure when conventional dilation fails.13 eus - td of pp is an attractive endoscopic approach in patients who have a small window of entry based on computed tomography (ct) findings, particularly in the case of lack of an endoscopically defined area of luminal bulging, in unusual locations of pps, with coagulopathy, with thrombocytopenia, with portal hypertension, with documented intervening vessels, in failed ctd or tpd and considering complication during ctd or tpd . A recent prospective randomized controlled trial regarding ctd versus eus - td revealed significant differences regarding technical success in treating pp.14 with regard to clinical outcomes (short - term and long - term results), however, there was no significant difference between ctd and eus - td.14 therefore, for luminal bulging pps, both ctd and eus - td can be selected and performed . However, for nonluminal bulging pps or if ctd or tpd has failed, eus - td has the theoretical advantage of reducing the risk of bleeding, perforation, and infection compared with ctd . The first meta - analysis comparing the technical success and clinical outcomes of eus - td and ctd for pps resulted in the same conclusion.15 utilizing eus - td for pp a prerequisite for eus - td is the presence of a well - defined mature wall . The fluid collection must be accessible endoscopically, such as being located within 1 cm of the gastric or duodenal walls; paracolic collections cannot be accessed and would require adjunctive methods such percutaneous drainage.16 thus, eus - td should be performed as a preferable approach to ctd or tpd (table 2). To date, current reports in the literature regarding eus - td for pp have documented recent developments and improvement of outcomes.17,18 currently, the type, size, and number of stents used for eus - td are the major concerns of interest . The fistula tract between the gastrointestinal tract and the pp is maintained with the placement of double pigtail plastic stents for preventing dislocation and migration . Although double pigtail plastic stents have been used to provide drainage, occlusion rates are high and endoscopic access to the pp cavity via the fistula is limited because of its small caliber . Therefore, placement of multiple small - caliber (including simultaneous placement of a pigtail stent and a nasocystic drainage catheter) (figs 13) or large - caliber pigtail plastic stents is required to maintain a large fistula for sufficient and effective drainage . However, small - caliber plastic stents are needed for multiple attempts and accesses to the cavity . These procedures may cause loss of the guidewire (failure of multiple stenting), proximal migration of the first stent into the cavity, additional time, and a more cumbersome procedure . On the other hand, large - caliber stents can be difficult to advance and deploy through the channel of the eus scope . Recently, tubular csemss (tcsems), which are used for the treatment of a biliary stricture, have been available for pp drainage instead of multiple plastic stents . The tcsems provide larger calibers than plastic stents, which might be advantageous for contaminated and excessive amounts of debris although it is much more expensive than plastic stents (table 3). The tcsems can also reduce the risk of perforation, leakage and bleeding because of minimal dilation and sealing of the fistula tract including tamponade effects . Several reports of a case or case series of pp have indicated the utility of csemss for drainage . A summary of these reports showed 93 patients with pp using csems (table 4).1933 the technical success rate from published cases was 100% (93/93 pps). Pp resolution was achieved in 94.6% (88/93 pps) with complete resolution in 90.6% (77/85 pps). The complication rate was 21.1% (19/76 pps). Among them, the most common complication was superinfection to pps, with a mild degree of severity . On the other hand, the csems migration rate was 3.9% (3/76 pps) and the buried csems rate was 2.6% (2/76 pps). Partial or full csems migration is a significant problem because csemss are tubular conduits and do not have anchoring flanges . To prevent migration, the placement of a double pigtail stent or a nasocystic catheter through the csems may be effective to serve as an anchoring effect . The currently available and used csemss were designed for drainage related to a luminal stricture, but were not related to a transluminal route . When a bile duct or an esophageal stent is used for pp, the longer protrusion on both the gastrointestinal tract and the pp cavity sides entails a risk of contact ulceration, bleeding, or migration . They are not good options in cases when the pp is not firmly attached to the gastrointestinal wall, because they do not apply any anchorage force and the risk of leakage is high.30 more recently, new dedicated anchoring fully covered semss (acsemss) for pp have been developed, such as wide flared end (fig . 4; (a, b) nagi stent, taewoong medical co., ltd ., seoul, korea,31 (c, d) bcf stent, m.i.tech co., ltd ., seoul, korea) or anchoring (fig . 4; (e) axios, xlumena inc ., mountain view, ca, usa)22 to prevent migration (figs 57). These types of acsems provide stent stability, minimize the risk of migration due to an anchoring effect, and maintain the larger sems lumen for passage, which may enable easy direct access into the pp cavity without a nonliquid component after expanding in full diameter . What is the optimal stent for pp? The answers to this question are straightforward . At the present shape22 is an ideal stent and is highly recommended for treating pp in terms of antimigration and the direct insertion of an endoscope through the acsems . The stent anchors are designed to distribute pressure evenly on the luminal wall and securely anchor the stent, thus preventing migration . The proximal and distal anchor flanges are designed to hold the bile duct and duodenal wall in apposition, preventing leakage between the two nonadherent organs . What remains controversial and yet to be determined are the appropriate period for stent placement and the optimal stent diameter . Stents for pfcs act as a conduit and facilitate drainage of pancreatic secretion from the disconnected gland . In a prospective randomized controlled trial involving the removal versus nonremoval of stents, the rate of pfc recurrence following stent removal was significantly higher, particularly in patients with main pancreatic duct rupture.34 it is likely that pfc resolution leads to the eventual adherence of the cavity wall, leading to the gradual migration of the stent toward the gastrointestinal lumen . Stent removal occurring before complete pfc collapse might lead to pfc recurrence, particularly if a communication exists between the pfc and the pancreatic duct.35 prolonged transluminal stent placement has been adopted as a strategy to prevent pfc recurrence, that is, the stent remaining in its proper position reduces the recurrence rate of pfc.36 on the contrary, the appropriate duration of stent placement is recommended to be short (7 to 10 days) because of a significant risk of stent migration if the stents were left in place longer than 10 days.37 however, the short duration of stent placement may not be sufficient to create an adequately mature fistula tract that will consequently tolerate balloon dilation and direct endoscopic necrosectomy.28 the clinical data on pancreatic abscess or infected won are more limited and generally poor, owing to the need to remove abscess and necrotic debris, than in the case of pp drainage . However, in collections with necrotic debris, the success rate falls with the drainage of cyst contents alone . Subsequent direct endoscopic necrosectomy has therefore been performed for an infected anc, pp, or won . We should consider direct endoscopic necrosectomy under the following conditions: 1) necrotizing pancreatitis is present; 2) us, eus, ct, or magnetic resonance images show solid components in the fluid collection; and 3) acute inflammation suggesting an infected won is present.38 several sessions are necessary for sufficient necrosectomy to improve inflammation . For this technique, placement of multiple plastic stents and repeated large - diameter balloon dilatation are required in each session . Recently, a prospective randomized controlled trial of direct endoscopic drainage / necrosectomy of pancreatic abscess or infected won versus surgical management has been performed.39 in this recent study involving patients with an infected won, endoscopic necrosectomy reduced the proinflammatory response (serum interleukin-6) as well as the new - onset multiple organ failure, intra - abdominal bleeding requiring intervention, enterocutaneous fistula or perforation of a visceral organ requiring intervention and pancreatic fistula compared with surgical necrosectomy . In the study design, multiple plastic stenting for infected won following repeated balloon dilation was performed . A summary of studies reporting the use of csems in 56 patients with pancreatic abscess or infected wons is shown in table 5.20,23,27,28,30,31,38,4043 the technical success rate (100%, 57/57 pancreatic abscess or wons) and the pancreatic abscess or infected won complete resolution rate (87.8%, 43/49 pancreatic abscess or infected wons) were high similarly to pps . The complication rate was low (9.5%, 4/42 pancreatic abscess or infected wons) compared with pps . Larger diameter csems without additional fistula tract dilation for the passage of a standard scope is needed to access and drain for pancreatic abscess or infected wons with solid debris . During direct endoscopic necrosectomy through the csems, such csems interferes with the operation of the endoscope . On the other hand, the sems length was selected on the basis of the size of the pp, pancreatic abscess or won, with 1/3 to 1/2 of the sems protruding into the gastrointestinal tract at the level of the flared ends permitting apposition of the pp, pancreatic abscess or won to the gastrointestinal tract.43 commercially available biliary semss neither offer a large diameter that allows a larger channel endoscope to be inserted in order to perform necrosectomy, nor permit complete apposition of the wons to the wall of the gastrointestinal tract . Therefore, an anchoring fcsems particularly with a dumbbell shape is also strongly desired for treating infected anc / pp or wons . The conventional single transluminal gateway drainage using transmural stenting (single or multiple plastic stents or large - bore semss) has allowed the complete resolution of unilocural or uncomplicated pfcs . However, single gateway drainage for complicated or infected wons is limited and often insufficient . Multilocular or huge infected won requires multiple transluminal gateway drainage because of the presence of undrained subcavities.4446 when subcavities or undrained areas of the main cavity are in a location far from the gastrointestinal tract, eus - td is not possible . Single transluminal gateway transcystic multiple drainage might be a better technique for these cases.45 if endoscopic intervention fails for complicated won, the hybrid technique using endoscopic and percutaneous approaches is recommended and might be a better approach . Eus - td with sems placement for infected pp, pancreatic abscess or wons is a technically feasible and apparently safe alternative to ctd and td . Eus - td with sems placement can be considered as the first - line therapy for pp . With increasing data showing better clinical outcome of eus - td with csems, it is highly recommendable to conduct a prospective randomized controlled trial of plastic stent versus csems for pp drainage to determine the long - term outcome and allow cost analysis . Finally, future clinical prospective studies should be conducted to validate local complications of acute pancreatitis on the basis of the revised atlanta classification.6
Scientists have recognized communication between the brain and the gut for more than 100 years, with studies in the early 19th and 20th centuries showing that a person s emotional state can alter the function of the gastrointestinal (gi) tract.13 one of the best examples is the work of william beaumont, an army surgeon, who became known as the father of gastric physiology . In the 1830s, beaumont, who was able to monitor gastric secretions through a fistula (a permanent opening in the stomach wall), noted an association between changing moods and gastric secretions . In the mid - to - late 1900s, research examining stress biology and its impact on human health uncovered clear connections between an individual s stress response and gut function . This classical view of top - down control that is, the brain s ability to control gut function is supported by evidence revealing that the brain influences body systems, including the gi tract, through neural connections of the autonomic nervous system and through humoral systems in the bloodstream . Both of these communication pathways are activated in stressful situations and influence gut function . What is exciting and new is the consideration of bottom - up control that is, how the gut, or more precisely the microbiota in the gi tract, can influence brain function . Researchers have recently shown that the presence of gut microbiota during early development influences the brain s neural connectivity related to anxiety and depression . Gut microbiota has been linked to behavior, to stress, and to stress - related diseases . Changes in gut microbiota may influence risk of disease, and manipulating microbiota may provide novel ways to intervene in clinical situations related to mood and anxiety disorders . Normal commensal microbiota colonizes the mammalian gut and other body surfaces shortly after birth and remains there throughout an individual s lifetime . In humans, the lower intestine contains 10 to 10 bacteria; that is, there are 10 to 100 times more bacteria in the gut than there are somatic cells in the human body.4 the interactions between commensal microbiota and its host are for the most part beneficial . In particular, the presence of commensal organisms is critical to immune function, nutrient processing, and other aspects of healthy physiology.5, 6 using the latest molecular and genetic tools, researchers have shown that several bacterial phyla are represented in the gut and that commensal populations show considerable diversity, with as many as 1,000 distinct bacterial species involved.7 in addition, factors such as genetics, age, sex, and diet continually influence the composition and profile of an individual s microbiota . 8, 9 in healthy people, there is considerable variability in gut bacterial composition, and yet if the same person s gut bacteria are examined at different times (a few months or more apart), they hardly change.4, 10, 12 but in stressful situations, or in response to physiological or diet changes, the microbiota profile may itself change, creating an imbalance in host - microbiota interactions, microbiota may influence the development of brain regions involved in our response to stress and control stress - related conditions such as anxiety and depression . In an attempt to understand these relationships, scientists manipulate gut bacteria in mice by raising germ - free mice in sterile isolators and then measuring the presence of gut bacteria . Much of this work draws upon standard animal behavioral tests that measure activity, approach, and avoidance . Mice have a natural tendency to explore their environment while avoiding open and brightly lit areas . The elevated - plus maze, a behavioral apparatus that is elevated aboveground (fig . 1), contains an area (with two closed and two open arms) for a mouse to explore . When a normal control mouse is placed in the maze, it tends to explore both arms but to spend most of its time in the closed one . When a germ - free mouse is removed from its sterile housing conditions and placed in the maze, it tends to spend significantly more time in the open arm . This suggests that mice without gut bacteria have low levels of anxiety - like behavior, since they spend more time in the aversive area of the testing apparatus.13, 14 another behavioral test uses the light - dark box, which has a dark, closed area connected to a light open area (fig . A normal control mouse explores both the light and the dark chambers with a preference for the darker one . However, germ - free mice spend more time in the light side of the apparatus, again demonstrating that mice without gut bacteria have low levels of anxiety - like behavior because the light chamber is considered the aversive region in this test, and germ - free mice spend more time in the light chamber.13, 15 germ - free mice have helped researchers explore whether there are particular times over a mouse s lifespan when microbiota - brain interactions are most important . Germ - free mice have been exposed to normal housing conditions at different times though their development . Exposure to normal housing conditions has revealed colonization of the sterile gi tract of germ - free mice with normal populations of gut bacteria . This also results in normalization of the undeveloped immune system that is present in germ - free mice . When adult germ - free mice were colonized with normal bacteria, they continued to show reduced anxiety - like behaviors, suggesting that the absence of gut bacteria early in development has a permanent effect on the brain wiring related to anxiety and exploratory behavior.14, 16 in contrast, when germ - free mice were colonized early in life as pups or during adolescence and then tested in adulthood, normal anxiety - like behavior was observed,13, 15 suggesting that microbiota influence the way the brain is wired early in development.17 in addition to studying mice, researchers have used antibiotic treatment to manipulate gut bacteria . Exposure to antibiotics in drinking water has been shown to lead to reduced numbers of gut bacteria in mice and to a reduction in the diversity of the bacterial population.18 consistent with work in germ - free mice, mice exposed to antibiotics for a single week showed increased exploratory behavior and reduced anxiety - like behavior, an observation that was linked to changes in the bacterial profile.19 two weeks following the end of the antibiotic treatment, both the bacterial profile and the behavior returned to normal, suggesting that transient changes in gut microbiota can influence behavior.19 having established connections among gut bacteria, the brain, and behavior, it is intriguing to consider the ways that microbiota may communicate with the brain . Certainly, classical thinking tells us that there are neural connections from the body to the brain through peripheral nerves, and, in particular, the vagus nerve, which provides information from the gut to the brain . Evidence that bacteria in the gi tract can activate the vagus connection to the brain comes from work showing that administering food - borne pathogens, such as citrobacter rodentium and campylobacter jejuni, to mice activated vagal pathways and related brain regions.20, 21 this neural activation occurred in the absence of a peripheral immune response, suggesting the presence of a direct link between the bacteria in the gut and the nervous system . In a recent study, feeding healthy mice probiotics, or good bacteria, decreased anxiety - like and depressive - like behaviors compared to control mice,22 while a related study showed that feeding mice probiotics activates neurons in the hypothalamus, a brain region known to play a role in stress reactivity.23 in the latter study, the activation of neurons in the hypothalamus was greater when mice were fed infectious bacteria leading to a robust peripheral immune response . This suggests that both peripheral nerves and blood - borne immune signaling molecules can contribute to gut - brain communication.23 at the level of the hypothalamus, the brain s autonomic nervous system control center, there is considerable evidence that psychological, physiological, and pathological challenges can activate the hypothalamus and turn on the body s stress response . It is fascinating that the communication pathways from gut microbiota to the brain also lead to activation of this key brain region . While the above - noted work establishes a neural pathway from the gut to the brain, a second important pathway for communication is the immune system . The immune system has two components, the innate immune system and the adaptive immune system . In germ - free mice, since gut microbiota are essential for immune system development, germ - free mice can be considered to have a low level of inflammation . When we consider the link between inflammation and anxiety - like behavior, we observe that a low inflammatory state is associated with low anxiety - like behavior, whereas higher levels of inflammation lead to increased anxiety - like behavior.17 for example, infection with the parasite trichuris muris in mice results in gut inflammation and increased anxiety - like behavior.24 in addition, chemically induced gut inflammation in an animal model of colitis also results in gut inflammation and increased anxiety - like behavior.24 evidence that the microbiota acts as a modulator of this immune - related increase in anxiety - like behavior is provided in the same reports stating that treatment with probiotic bifidobacterium longum alleviated the anxiety - like behavior.24,25 these observations suggest that probiotic treatment may have potential for treatment of inflammation and related anxiety symptoms . Commensal bacteria play an important role in maintaining the integrity of the intestinal tract, and in situations of stress or disease, increased intestinal permeability can contribute to increased inflammation.26, 27 increased intestinal permeability, sometimes referred to as leaky gut, can lead to translocation of bacteria out of the lumen of the gi tract across the intestinal layer . This is an additional pathway that microbiota and pathogenic bacteria use to communicate with the brain via the immune system or through activation of local neurons in the enteric nervous system (ens). The ens is a part of the autonomic nervous system that is housed in the gut and is responsible for gut motility and other normal gut functions.28 it is a vast network of neurons that are the first points of contact for microbiota in the intestinal lumen and are an important component of the brain - gut axis . One of the most common clinical features of depression is dysregulation of the stress response system, the hypothalamic - pituitary - adrenal (hpa) axis.29 as was previously noted, in response to psychological, physiological, and pathological challenges, neurons in the hypothalamus are activated and signal the pituitary to release adrenocorticotrophic hormone into the bloodstream, which in turn activates the adrenal gland to release the stress hormone cortisol . The stress response, or hpa activation, is part of our normal homeostatic processes, and yet, in depression, it is often overactive or, in some cases, underactive.29 one of the first studies considering stress and microbiota demonstrated that germ - free mice have an overactive stress response.23 a more recent study has shown that stress exposure during early life in rats disrupts the microbiota profile and leads to increased stress reactivity in adulthood.30 importantly, in this study, treatment of rat pups with probiotic lactobacillus sp . Normalized stress hormone levels.30 early - life stress also leads to increased depressive - like behavior in adult rats, and a similar study showed that treatment of rats exposed to stress during early life with the probiotic bifiodo infantis reduced the depressive - like symptoms in adulthood.31 overall these recent studies imply a link among microbiota imbalance, stress - related behaviors, and stress reactivity, and also suggest that probiotic treatment may be a good approach to treating stress - related symptoms . To date, researchers have done little work related to stress and microbiota in humans, and in particular, there have been no studies that directly link microbiota to depression or anxiety . The most promising of the clinically related work shows that probiotic administration in people may have antidepressant or anxiety - reducing effects . In one study, researchers used several questionnaires to test the effects of probiotics on stress, anxiety, depression, and coping . Their results showed that the probiotics group had less psychological stress than the control group did.32 in a separate study, researchers were able to show that healthy people with low mood at the beginning of the study showed improvement in mood following probiotic administration for three weeks.33 finally, in a clinical study on individuals with chronic fatigue syndrome, administration of probiotics over a two - month trial resulted in fewer anxiety - related symptoms.34 these studies show that clinical trials to date support a role for microbiota in anxiety and depression, and also demonstrate the potential for treatment with probiotics . There is no doubt that research in the last decade has established a link between gut microbiota and brain function in mice . We have learned several things: (1) gut microbiota are a large population that is important to healthy metabolism and brain function, (2) gut - brain communication pathways include neural connections, (3) gut microbiota are significant during early development and can influence the wiring of stress circuitry in the brain, and (4) probiotics, or good bacteria, have been shown in animal and human studies to have a beneficial impact on mood symptoms . While these findings are both exciting and promising, we should not make the mistake of thinking that we have found the answers to all clinical situations related to mood . Microbiota certainly an important modulator of health must be considered as one component of a complex, multifaceted communication system needed to establish a healthy balance for brain development and function.
Pulmonary adenocarcinoma is a common malignancy that is prone to calcification, although bone formation in primary lung adenocarcinoma is extremely rare . Heterotopic ossification in primary lung adenocarcinoma was first described by mclendon et al . In 1985 (1). To our knowledge, however, only 11 cases of pulmonary adenocarcinoma with heterotopic ossification have been reported in the literature, and the isolated cases described in these reports involved ossification in bronchogenic carcinoma (1 - 11)., we describe the clinicopathologic features of ten cases of pulmonary adenocarcinoma with heterotopic ossification in addition to a review of previously published data and a discussion on the histogenesis of bone formation . Ten cases of pulmonary adenocarcinoma with heterotopic ossification diagnosed in the department of pathology at the samsung medical center in seoul, korea, between 2003 and 2007 were considered (table 1). Hematoxylin and eosin (h&e)-stained slides were then prepared for analysis by two experienced pulmonary pathologists to confirm the diagnosis and to assess multiple histologic features . Immunohistochemical staining was performed in patients 1 - 8 using 4-m - thick tissue sections and a bond polymer intense detection system (visionbiosystems, vic, australia) according to the manufacturer's instructions with minor modifications . In brief, each formalin - fixed and paraffin - embedded section was deparaffinized with bond dewax solution (visionbiosystems), and subjected to antigen retrieval using bond er solution (visionbiosystems) at 100 for 30 min . The sections were then incubated for 15 min at room temperate with thyroid transcription factor-1 (ttf-1) (8g7g3/1; 1:3,000; dako, glostrup, denmark), p53 (do-7; 1:1,000; novocastra, newcastle upon tyne, u.k . ), ki-67 (mib-1; 1:500; dako), osteopontin (op3n; 1:400; novocastra), osteocalcin (fl-100; 1:50; santa cruz biotechnology, santa cruz, ca, u.s.a . ), runx-2 (1d2; 1: 1,000; abnova, taipei, taiwan) and transforming growth factor-1 (tgf-1) (rabbit polyclonal; 1:25; neomarkers, fremont, ca, u.s.a .) Using a biotin - free polymeric horseradish peroxidase - linker antibody conjugate system in a bond - max automatic slide stainer (visionbiosystems), and visualized using a 3,3'-diaminobenzidine (dab) solution (1 mm dab, 50 mm tris - hcl, ph 7.6, and 0.006% h2o2). Formalin - fixed paraffin - embedded human gallbladder tissue for osteopontin, kidney for osteocalcin, and placenta for runx2 and tgf-1 were used as positive controls . The intensity of cytoplasmic staining for osteopontin, osteocalcin and tgf-1 and nuclear staining for runx2 was scored as follows; 0 (negative), + (weak), 2 + (moderate), and 3 + (strong). The signal was also regarded as positive for osteopontin, osteocalcin, runx2, tgf-1, ttf-1, and p53 if more than 10% of the specific cell population was immunostained . Chi - square and fisher exact tests were used to evaluate the association between the expression of tgf-1, osteopontin, osteocalcin and runx2 for each the tumor and the fibroblastic stroma . All analyses were performed with spss software (version 15.00, spss, chicago, il, u.s.a . ). Chi - square and fisher exact tests were used to evaluate the association between the expression of tgf-1, osteopontin, osteocalcin and runx2 for each the tumor and the fibroblastic stroma . Statistical significance in this study was considered at p<0.05 . All reported p values all analyses were performed with spss software (version 15.00, spss, chicago, il, u.s.a . ). The clinical features of ten cases of pulmonary adenocarcinoma with heterotopic ossification are listed in table 1 . The age of the patients ranged from 57 to 70 yr (mean 62.4 yr), and the male: female ratio was 4:6 . Eight patients were non - smokers, while the remaining two (patients 2 and 5) had stopped smoking 10 and 15 yr earlier, respectively . Radiologically, radiopaque shadows in the center of the lung mass indicating calcification or ossification were found in nine of ten patients (all except patient 1). Due to the high mineral densities a trabecular pattern was observed in the center of the lung mass in patient 2, suggesting intratumoral ossification (fig . Nine of the patients received lobectomies while patient 2 received a bilobectomy, including right upper and middle lobes with dissection of the mediastinal lymph nodes . Neoadjuvant chemotherapy and concurrent chemoradiotherapy were administered in patient 6 and 7, respectively, while adjuvant chemotherapy was administered in patient 9 . The tumor, which initially appeared to be solid gray - white solid masses, had a granular appearance during sectioning, indicating calcification or ossification . The tumors, which ranged in diameter from 2 to 7 cm (mean 3.8 cm), were located in the right upper lobe in four patients, the left lower lobe in three patients, the right lower lobe in two patients, and in the right middle lobe in one patient . Histologically, the tumor pattern was papillary - tubular in eight patients, papillary in one patient (patient 3), and solid - tubular pattern in one patient (patient 6). The tumors were moderately differentiated in all cases except case 6, which exhibited poor differentiation . Mitotic figures were frequently observed in the tumor cells, and all of the tumors contained fragments of osseous tissue within the abundant central fibroblastic stroma (fig . Dense connective tissue was present in the area of ossification, but was absent from the periphery of the cancer (where ossification was not observed). In patient 8, occasional psammoma bodies were observed in the tumor papillae . Cellular atypia was absent from the stromal fibroblastic cells, osteoblasts, and osteocytes (fig . Minimal tumor necrosis (<5% of the tumor area) was present in patients 2, 6, 7, and 8; however, no necrosis was found in the ossification area . In patient 1, chronic inflammation was detected in the fibroblastic stroma; however, none of the other patients exhibited a mucin pool, inflammation, or preexisting calcification of the fibrous stroma in the area of ossification . The size of the ossification area ranged from 0.05 to 2 cm (mean 0.78 cm). A tiny bony trabecula measuring 0.05 cm in length was observed in patient 1 . Within the osseous tissue, osteoid formation with calcification and fully mature trabecular bone having osteocytes, osteoblastic rimming, and lamellation were observed . Lamellation of the trabecular bone under polarizing light was observed in eight of ten patients (fig . 4), while rows of osteoblasts were seen along the edge of the osseous trabeculae, in all ten patients . Evident marrow spaces were observed in six of ten patients, while osteoclastic activity suggesting bone remodeling was found in five of ten patients . Metastasis of the peribronchial lymph nodes (n1 nodal stage) in patients 2 and 6, and the mediastinal (n2) lymph nodes in patient 7 was present, but bone formation was not detected . The tumor and fibroblastic stromal cells reacted with antibodies against tgf-1, osteopontin, osteocalcin and runx2 (fig . The intensity of tgf-1 and runx2 staining was the same in the tumor and fibroblastic stromal cells . The immunoreactivity against osteopontin and osteocalcin was the stronger in the tumor and the fibroblastic stromal cells, respectively . The intensity of osteopontin was more significantly strong in the fibroblastic stroma of ossification area compared to the non - ossification (p=0.029). However, there was no significant difference in those of osteocalcin (p=0.097). Positive collagen type iv immunostaining was observed in patient 3 in the fibroblastic stromal cells . Positive reactivity for p53 was observed in six of eight patients (75%), and the index of positivity in the tumor cells ranged from 30 to 90% (mean 55.8%). The ki-67 labeled proliferating index value for the tumor cells ranged from 1 to 35% (mean 11.3%); however, in the fibroblastic stromal cells, the index value was 0% in all cases . All ten patients were alive during the follow - up period (4 - 52 months, mean 22.8 months). Two patients (patients 2 and 3) had recurrent remnants in the right lower lobe and at the lobectomy stump area 16 and 30 months after surgery, respectively, and chemotherapy was administered . Intratumoral calcification has been observed in various neoplasms . In fact, benign heterotopic bone formation within neoplasms has been sporadically reported in the kidney, liver, gastrointestinal tract, breast, thyroid, skin, and soft tissues . In contrast to dystrophic calcification, which frequently occurs in necrotic areas of cancer, heterotopic ossification within primary lung carcinomas is extremely rare . Although bronchial carcinoid is one such calcifying or ossifying tumor, a review of the literature revealed only 11 other cases of heterotopic ossification in pulmonary adenocarcinoma and two cases in pulmonary squamous cell carcinoma (1 - 13). All previously published cases of pulmonary adenocarcinoma with heterotopic ossification are listed in table 4 . In those cases, the patients ranged in age from 49 to 76 (mean 62.5 yr) and the male: female ratio was 7:4 . The predominant histologic type of the tumors was moderately differentiated adenocarcinoma with a dense fibrous stroma located at the peripheral lung zone with pleural dimpling . Although intrapulmonary calcification is frequently observed radiologically, distinguishing calcification from ossification is difficult . In some cases in our series however, ossification can be differentiated radiologically from calcification when a trabecular or reticular pattern is detected by ct (14, 15). This is a common process that occurs under a variety of pathologic conditions, such as hypercalcemia, necrosis, extracellular mucin and chronic inflammation . Several theories exist regarding the mechanism of intratumoral calcification and ossification, including calcification in the presence of degenerative or necrotic tissues (dystrophic calcification), entrapment of preexisting calcified scar tissue or granulomatous disease, and calcium deposition within the tumor as a result of mucus production by tumor cells (16, 17). Both processes feature the transformation of a primary trabecular network into mature bone, but differ in terms of the starting point . Intramembranous bone formation involves the transformation of a mesenchymal template into bone, while endochondral ossification involves the replacement of a preexisting hyaline cartilage template into bone . The sequence of events in intramembranous bone formation includes the formation of mesenchymal condensations via a process controlled by wnt, hedgehog, fibroblast growth factor, and tgf- family polypeptides; differentiation of the mesenchymal cells into osteoblasts, deposition of the bone matrix by the osteoblasts, mineralization and differentiation of osteocytes by interstitial growth under the control of the transcription factors cbfa1/runx2 and osterix . Cbfa1/runx2 is the earliest and more specific indicator of osteogenesis and its expression is induced by bone morphogenic protein 7, followed by the expression of osteocalcin and osteopontin . Osteopontin and osteocalcin are non - collagenous multifunctional glycoproteins routinely found in mineralized tissue, where it is believed to play an integral role in the cellular response to mechanical stimuli . These proteins also play a role in the initial formation of the matrix, depending on the mode of ossification and bone morphogenesis (18, 19). A progressive increase in the amount of osteopontin in the membranous bone matrix during the maturation of new bone was previously reported (20). Osteocalcin is a specific secretory protein expressed only in terminally differentiated osteoblasts under the control of cbfa1/runx2 . We propose that the heterotopic ossification of primary pulmonary adenocarcinomas occurs via intramembranous bone formation, and is induced by the tumor cells . Our reasoning is as follows; no necrosis, mucin pool, or inflammation of the fibroblastic stroma in the area of ossification was observed in any of the patients, except patient 1, who exhibited chronic inflammation of the fibroblastic stroma; preexisting calcification with hyaline cartilage formation or a bluish chondroid stroma was not observed in the areas of ossification; the level of immunoreactivity against tgf-1, osteopontin, osteocalcin and runx2 was highest in the tumor cells and the fibroblastic stroma; and osteopontin was more expressed in fibroblatic stroma surrounding the area of ossification compared to the area of non - ossification, which indicate osteopontin has more osteoblastic activity in fibroblastic stroma surrounding the area of bony trabecullae . Still, we cannot completely exclude the effect of the neoadjuvant chemotherapy applied to patient 6 and neoadjuvant concurrent chemoradiotherapy to patient 7, and patient 1 exhibited chronic inflammation of the fibroblastic stroma and a tiny bony trabecula . However, none of the other patients showed necrosis with cholesterol granuloma or inflammation, the aggregation of foamy macrophages, in the area of ossification . The stromal response should be differentiated from true sarcomatous participation in the tumors . Pulmonary carcinosarcomas show a distinct malignant stroma and osteoid formation . As far as the present tumors are concerned, the possibility of a carcinosarcoma is ruled out by the lack of malignancy in the fibroblastic stroma and osteoid . The observed intratumoral ossification consisted of fully matured bone elements with no cellular atypia, and mitosis was not detected in the stromal fibroblastic cells . Additionally, the ki-67 labeled proliferating index value was 0% in the stromal fibroblastic cells and bony trabeculae . Therefore, we presume that the osseous elements were produced through benign reactive or metaplastic processes . In conclusion although, distinguishing calcification from ossification radiologically is difficult, ossification can be recognized based on the presence of a trabecular or reticular pattern . We detected immunoreactivity against tgf-1, osteopontin, osteocalcin and runx2 in the tumor cells and fibroblastic stroma surrounding the area of ossification . Our results indicate that heterotopic ossification in primary pulmonary adenocarcinoma occurs via an intramembranous bone formation and osteopontin has more osteoblastic activity in fibroblastic stroma surrounding the area of bony trabecullae . However, additional studies with a greater number of cases are needed to elucidate the mechanisms of intratumoral osteogenesis and osteogenic metastasis and their prognosis.
The classification of orbital tumors is very difficult because tissues of various origins and structures are present in this region [13]. The most frequently used classification divides tumors into various categories, including: primary tumors deriving from the orbital tissues, intraocular, corneal, conjunctival tumors, and tumors penetrating the orbital cavity from adjacent areas, as well as metastases from other areas of the body . The topographic anatomy of the eye socket surrounded by paranasal sinuses and the cranial cavity, and the resulting mutual penetration of disease processes in that region makes the eye socket an interdisciplinary region [68]. Therefore, the orbital cavity is an area of interest of many specialists, including ophthalmologists, ent (ear, nose, and throat) doctors, endocrinologists, neurosurgeons, plastic surgeons, and maxillo - facial surgeons, and the application of many diagnostic methods is needed [7,911]. The diagnostics and treatment of the proliferation processes in the eye socket require the close co - operation of the above - mentioned specialists . The main manifestation is the protrusion of the eyeball and limitation of eyeball movement, and dysopsia, swelling, and reddening of conjunctivae, as well as blepharo - edema . The aim of the study was to perform a histo - clinical analysis of patients with primary orbital tumors, together with the analysis of localization of the tumors within the eye socket, their extensiveness, and relation to the adjacent structures, possible penetration to paranasal sinuses, and estimation of long - term results of treatment . The study was conducted on a group of 122 patients with primary orbital tumors, undergoing surgical treatment in the ent department of the medical university of silesia in katowice, poland, in the years 19902013, of whom 68 were women (55.7%) aged 2379 years (average age: 45 years) and 54 were men (44.3%) aged 2772 years (average age: 40 years). The tumor was located in the left eye socket in 58.2% of patients and in the right in 41.8% . The characteristics of patients, depending upon the histopathological diagnosis, are presented in tables 1 and 2 . In the course of pre - operative diagnostics, most patients had the following examinations performed: x - ray imaging, ct (computed tomography), and/or mri (magnetic resonance imaging). The locations of tumors were divided into 4 areas: upper - lateral, upper - medial, lower - lateral, and lower - medial on the basis of ct or mri findings (table 3). Figure 1a and 1b present results of ct examination of a 65-year - old woman with carcinoma plano epitheliale of the right orbit . Figure 2 presents mri results of a 29-year - old woman with pleomorphic adenoma in the left orbit . Each patient with the diagnosis of orbital tumor underwent a full ophthalmological examination, before and after the surgery . The examination comprised assessment of vision acuity, assessment of the protrusion degree, assessment of the field of vision, examination of the fundus of the eye, and assessment of eyeball mobility . In some patients, fine - needle aspiration (fna) biopsy of the orbital tumor was performed before the operation . However, because of the specific topographic relations in the eye socket, fna is difficult and has a high percentage of complications and false - negative results, especially in case of intra - conus location of the tumor . All patients analyzed in this study underwent surgical treatment . In a case of malignant tumors (with the exclusion of lymphomas) the orbital exenteration was performed, followed by complementary radio- and chemotherapy . In cases in which the post - surgical histopathological examination revealed a malignant lymphoma texture, in - depth diagnostics was performed, and after it was confirmed that the focus in the eye socket was the only one in the body, patients underwent hospital hematological treatment . In patients for whom histopathologic diagnosis of benign tumor was confirmed, depending on the tumor location, 1 of the 2 types of orbitotomy was performed . Lateral orbitotomy according to krnlein - reese - berke was performed each time in a case of non - malignant tumors located laterally in relation to the optical nerve, whereas medial orbitotomy according to sewall was performed in case the non - malignant tumors located in medial quadrants of the eye socket . The post - operative material was assessed each time by a specialist in the field of histopathology . In a case of malignant tumors, all patients were strictly followed up in the laryngological outpatient clinic that is connected with the ent department . The study protocol was approved by the local research ethics committee of the medical university of silesia in katowice . Descriptive statistics and chi - square test () were used for data analysis, using spss 17.0 software . Among 122 patients with primary orbital tumors, 58.2% had the tumor located in the left eye socket, while 41.8% were in the right one (figure 3). The most frequent manifestations in patients with primary tumors of the orbital cavity were as follows: protrusion of the eyeball (displaced at least 2 mm) occurred in 100% of patients, limitation of eyeball movement was observed in 45%, dysopsia (diplopia in 16%) visual acuity attenuation occurred in 43%, pain in the eyeball in 30%, swelling and reddening of conjunctivae and eyelids in 54%, headaches in 26%, blindness in 6%, and blepharophimosis in 51% . Regarding the type of orbital tumors, malignant tumors were observed in 45.9% of patients and non - malignant tumors were diagnosed in 34.4% of patients . Sarcoidosis, amyloidosis, or mikulicz s disease tumors were very rare (table 2). In analyzing the frequency of occurrence of various histopathological types of orbital tumors, malignant ones were found to be statistically significantly more frequent than benign neoplasmatic (p<0.001) or inflammatory tumors (p=0.006). Among 56 patients with malignant tumors operated on in our ent department, 13 cases had stage t1, 27 had t2, 12 had t3, and 4 cases of tumors were operated on in stage t4 . Metastases to lymph nodes of the neck region (stage n1) were diagnosed in 4 patients . Every time the location of the tumor in the particular quadrant of the eye socket was assessed, the estimation also determined eventual penetration of the tumor outside the eye socket area (table 3). The analysis of tumor localization inside the orbital cavity revealed that 27.87% of all tumors were situated in the lower - medial part of orbital cavity, 25.4% in upper - lateral and lower - lateral, and 19.67% cases were localized in the upper - medial part (table 3). Statistical analysis indicated that tumors simultaneously occupying 2 localizations upper - lateral and upper - medial are statistically significantly rarer than others . There were no statistically significant differences between the following localizations: lower - medial, upper - lateral, and upper - medial (p=0.331). Tumors were observed statistically significantly in lower - medial in comparison to upper medial localization (p<0.001), upper - lateral and upper - medial (p=0.006), and upper - lateral (p=0.09). Extension to paranasal sinuses was found in 23 patients (frontal sinus 8 cases, ethmoidal sinus 6, maxillary sinus 9). Figure 4 presents ct examination (3d reconstruction) with malignant tumor (carcinoma anaplasticum) of the upper - medial part of the right orbit penetrating to the cranial cavity . The complications after surgical treatment of benign and malignant orbital tumors are divided into vascular, neuro - muscular, inflammatory, and others (table 4). The most frequent complication after lateral orbitomy was paralysis of muscles responsible for eyeball movement (8.8%). The most frequent complication following medial orbitomy was paralysis of muscles responsible for eyeball movement (15.6%). After orbital exenteration, the most frequent consequence was hemorrhage (8.9%) (table 4). Among 122 patients with orbital tumor, biopsy was performed in 59 cases (48.4%). In 23 patients (38.9%) the results of histopathologic examination and fna biopsy differed from each other . In 20 cases (16.9%) that discrepancy concerned the pathomorphological texture, which did not require reoperation of patients . In the other 3 cases (5.1%) the discrepancy of examination results concerned the type of tumor (benign / malignant). In those patients, the histopathological examination confirmed the diagnosis of malignant tumor, which was not demonstrated by fna before the surgery, and for that reason reoperation was necessary for the procedure to be radical . Vision acuity after specific orbitotomy procedures was checked by an ophthalmologist in each patient at 1 and 3 months and 1 year after the operation . The operation performed in patients with benign tumors did not cause a deterioration of visual acuity in a comparison to before the operation . Each patient had an obligatory ophthalmological examination after the operation . In patients with inflammatory tumors of the orbital cavity (19 cases), a temporary deterioration of visual acuity, lasting up to 3 months after the operation, was observed in 4 individuals, whereas a permanent deterioration of visual acuity of 1 diopter was diagnosed in 6 patients . When cosmetic defects were involved, a linear scarf after a lateral orbitotomy performed by krnlein - reese - berke method reaching a lateral eye angle was not a serious problem for men, but 9 women treated it as a cosmetic defect despite the fact that it could be partially covered by either by glasses or by an eyebrow in the case of horizontal orbitotomy . Of the total number of 56 cases of malignant tumors, local recurrence up to 5 years was found in 36 (64.3%) cases . All those patients died . In the other 20 (35.7%) cases of malignant tumors, the patients remained under close follow - up in the outpatient clinic, without signs of local recurrence (follow - up from 1 to 17 years). Primary orbital tumors are very rare . According to the american cancer society, frequency of incidence of orbital tumors is less than 1 for 100 000 persons . The general symptomatology of developing orbital tumors is initially quite poor, because surrounding tissues adjust to the slowly growing tumor, delaying definitive diagnosis . Only after it reaches about 1 cm does the tumor begin to push out the eyeball, and the patient has the vague sensation of tension and bursting in the eye socket . As the tumor enlarges, protrusion of the eyeball occurs, along with limitation of eyeball movement, and dysopsia . Tumors located in the orbital conus cause a relatively quickly increasing protrusion of eyeballs, with deteriorated mobility of the eyeball and early changes at the fundus of the eye and dysopsia . The selection of surgical method for the treatment of primary orbital tumors always depends upon the type of pathological texture of the tumor, its location within the eye socket (in relation to the eyeball and the optic nerve), and directions of its possible penetration . In malignant tumors, the method of choice is orbital exenteration, possibly followed by radio- and chemotherapy . In non - malignant tumors, on the other hand, depending upon the primary location of the tumor, the approach is performed through the medial wall (sewall s approach), through the upper wall that is the ceiling of the orbital cavity (dandy s and naffziger s approach), through the lower wall (hirsch s method, with approach through the maxillary sinus), via the eye socket (knapp s approach), and via the lateral wall (kronlein s approach). The lateral subtemporal approach, developed in 1888 by kronlein, consists of temporary osteoplastic resection of the eye socket lateral wall . All orbitotomies can be divided into simple ones without mobilization of bones and those with engagement of bones (with mobilization). In our material, on postoperative histopathological examination, the most frequently occurring tumors of the eye socket were pseudotumor inflammatorius (14.75%) and lymphoma malignum (11.47%). In the material analyzed by piekowski et al ., malignant neoplasms of the eye socket occurred in 24% of the patients, whereas inflammatory lesions occurred in 4% . According to other authors, the most frequent eye socket neoplasms are inflammatory pseudotumors . Lateral orbitotomy has been the most frequent method of tumor resection . In caucasian populations, the percentage of people with malignant lymphoma is 515% . In the elderly population (over 60 years of age), it occurred less frequently, in only 3 patients . In the studies of li et al . And concerning orbital tumors, non - malignant tumors occurred most frequently in that area of the facial skeleton, whereas malignant tumors occurred considerably less frequently . On the other hand, shinder et al . Demonstrated that in patients with orbital tumors, primary orbital tumors were the most frequently occurring ones (64%) a substantial percentage of orbital tumors comprised malignant neoplasms . Assessing the tumor location in our material, we detected such tumors most frequently in the inferior medial quadrant . In the studies of other authors, frequent locations of orbital lesions included he superior half of the orbit, the anterior orbit, and the extraconal space . Many studies of primary orbital tumors have demonstrated a relationship between the types of orbital tumor patients and their age at diagnosis, but these relationships have not yet been clarified [1820]. Our research confirms the observation that the complex nature of orbital pathologies may be the cause of diagnostic and therapeutic difficulties . The basic method of treatment of malignant orbital tumors is surgery combined with irradiation and/or chemotherapy . Late diagnosis is decisive for radical surgical procedures to be performed, which drastically reduce the quality of life and chances for complete recovery . Fine - needle aspiration (fna) of the orbital tumor is an important diagnostic examination, and in case there are not contraindications it should be performed each time an orbital tumor is diagnosed . The results of treatment of malignant orbital tumors are not satisfactory . In the analyzed group of 56 patients with malignant tumors, the tumor location, histopathological diagnosis, and postoperative complications provide us with important information for the diagnosis of type of tumor prior to biopsy or tumor resection and for the determination of the treatment strategy.
There are many challenges in reproductive health and it is necessary to train experts to manage them . The aim of this study was to define the tasks of master of science (msc) graduates in reproductive health through comprehensive needs assessment to establish the course . The study comprised of three steps . In the first step, through literature review, the draft and basic fields of main tasks were defined . In the second step, by establishing a focus group of 10 experts, the tasks were extracted on the basis of the country s needs . In the third step, a delphi study was carried out among 51 experts who were selected to finalize the list of tasks and their priorities using three criteria of importance, feasibility and availability . 57 tasks were extracted with regard to the four main functions of management and planning, education, consultation, and screening in reproduction age of men and women . According to delphi s results and their priorities, 45 tasks were important and feasible but not available, and they were higher - priority tasks . The tasks extracted are consistent with the framework of reproductive health provided by the world health organization (who) and the american guideline of educational planning . However, considering the differences of problems in iran comparing with other countries, the list is not exactly similar to any list prepared for other countries . Therefore, it is necessary to consider the results of this research in university curriculums . Reproductive health is an important health topic, adolescence, and puberty of men and women . It also influences the health of future generations . By expansion of science, particularly medical sciences, and considering the importance of health, reproductive health is one of the appropriate indices in evaluation of countries development, especially for developing countries . According to the definition provided by who, reproductive health is a state of complete physical, mental, and social wellbeing in reproduction process and function throughout one s life . Therefore, all people should have a healthy and satisfactory sexual life, and be able to freely make decision about the time and method of bearing their child . In this regard, all people should have the right to access information, facilities and the highest standards of reproductive and sexual health without any discrimination, obligation or violence . Contrary to the definition of reproductive health and the policies of the united nations population fund, most countries, including the usa, canada, and japan, mostly rely on the traditional approach of mother and child health, based on the susceptibility of women in reproduction and fertility roles and paying special attention to the physiologic characteristics of women and their needs . In this regard, ministry of health is responsible for policy - making and general planning for men and women in various aspects . It should be noted that in recent years, application of the gender equality policies and the working plan of the women s world conference in beijing have caused considerable development in health sector and the policies and plans of ministry of health on topics, such as sexual health and aids, screening of cancers, mental health, and prevention of domestic violence . Our society also faces some challenges in reproductive health, which should be fundamentally addressed . Moreover, by following the health and education regulations, these problems can be prevented or treated, or their rehabilitation be facilitated . Some of these problems to consider are infertility, breast cancer, prostate cancer, puberty health, sexual dysfunction, illegal abortions, menopause, health care in these stages of life and new fatal sexually transmitted diseases, such as aids . For instance, breast cancer is among the most common cancers in iranian women . By increasing the general knowledge, timely diagnosis and effective treatment, more than 50% of the cancer patients would experience a long life . Therefore, organized educational, health, and therapeutic plans on prevention of breast cancer are required to increase the knowledge level and attitude of women toward breast cancer, and consequently promoting their participation in the screenings . With regard to abortion, the statistics published in 2008 indicated that the global rate of unsafe abortion increased from 19.7 million cases in 2003 to 21.8 million cases in 2008, which mostly occur in third world countries . This is while the rate was expected to reduce, owing to the improvement in health facilities and conditions ., almost one fourth of iranian women experience primary infertility in their married life, with the incidence rate of 3.4% . The couples age, health, treatment modalities, the age of marriage, and fertility potential are important factors in interpretation of the prevalence of primary infertility . By providing training and access to appropriate consultation considering the problems exist in sexual and reproductive health, it is necessary to establish an applied major, which directly deals with the problems of reproduction in the society . In this regard, defining the tasks of graduates in this major in iran as the first step of educational planning is necessary . Occupational analysis is usually the first step in educational planning to determine what students should learn . Occupational analysis of educational planning is fragmentation of the learning items that the students are expected to learn about how to do the job . Occupational analysis helps us to define the skills required for students in learning the prerequisites . Therefore, it is necessary to consult the specialist in the field and those who may involve in the process in this respect . The aim of this study was to define the tasks of the msc graduates of reproductive health by experts . Web services have been used in some vast needs assessment programs in the world . Considering the extension and distribution of the study population, this method was more appropriate for the study . The study comprised of three steps . In the first step, through literature review, the draft and basic fields of main tasks related to the job of msc graduates of reproductive health were defined . Then, in the second step and by establishing a focus group of 10 experts of various specialties, the fields and tasks were extracted on the basis of the country s needs . The group consisted of two faculty members of midwifery, four phd students of reproductive health with the career in being faculty members, two obstetricians, one geneticist and one urologist . The group members had a history of working in this field and some of them were involved in curriculum planning . Then, small groups of three experts were formed to discuss the list of the major s establishment objectives and its tasks, and finally they achieved consensus on the items . To make sure about the tasks that were extracted by the focus group of experts, the tasks were sent for the representative of each group, and some required modifications were done and returned back to them; ultimately the approved tasks were determined to be used for opinion poll of other experts . In the third step, delphi study was carried out using the web to finalize the list of tasks and their priorities . At this step, it was necessary to perform priority setting of the tasks and the opinions of experts were obtained . The sampling was goal oriented, and the experts were selected among the iranian obstetrics and gynecology specialists, phd students of reproductive health, the professionals in the field, faculty members of reproductive health, mother and child health, and midwifery . The faculty members were selected from the medical universities with msc courses, such as isfahan, ahwaz, tabriz, tehran, shiraz, and mashhad, and the medical universities which served as the referral centers of some provinces, including yazd, kermanshah, mazandaran, golestan, guilan, and sistan - va baluchestan . The aims of the study and the method of opinion poll were described on a website . The questionnaire included the items on tasks extracted in previous steps, which were presented along with the opinion poll guideline and priority - setting criteria . Furthermore, the website guideline with illustrations and examples was prepared and mailed to the participants . To avoid problems related to it priority setting was performed according to a method of altschuld s book, with the three criteria of importance, feasibility was defined as the level the task could be fulfilled by a reproductive health msc graduate and availability was the fact that this service is now provided or not . Availability was scored in a 1 - 3 range; if the service is now completely provided, it scored 3, if it is not provided at all, it scored 1, and if it is provided to some extent, it scored 2 . Accordingly, the importance factor was scored in a 1 - 5 range, and the feasibility was scored between 1 and 3, and the participants scored the items according to the lowest and highest scores . The items that obtained scores less than the median value (3 for 5-point scale and 2 for 3-point scale) were considered to be less important or unavailable or infeasible all priority conditions of the tasks in the opinion poll webpage, the participants could write down their comments on each task in specific boxes . The comments were reachable for the researcher as they were sent . After performing the opinion poll, the scores of each priority - setting criterion and the mean score for each task were calculated . In the first step, the draft of tasks in 19 main areas was obtained using literature review . Then, in the second step, the tasks were completed and modified by evaluation of the focus group . Then they were categorized on the basis of the four main stages of reproduction and its four subsidiary classifications . 57 tasks were extracted with regard to the four main functions of management and planning, education, consultation, and screening in reproduction age of men and women; that is, before and during puberty, before marriage, reproduction (pre- pregnancy, pregnancy, post - pregnancy and infertility), and menopause and older age . In the third step, to achieve the best possible results from the opinion poll, we tried to make use of the comments of experts and health managers from ministry of health and different universities, who were scientifically competent and had enough experience in this field . Of those who attended the study, 33% were specialists in the field of mother and child health, and 29% in the field of reproductive health . The 57 tasks and their mean scores assigned to priority- setting criteria are provided in table 2 . Results of priority setting of the tasks with regard to table 2, tasks no . 18, 22, 26, 35, 36, 37, 38, 41, 42, 43, and 44 obtained the mean importance score above the median score 3, the feasibility score above the median score 2, and the availability scores above the median score 2 . This shows that the item is important, feasible, and available, and further action is not required for it; thus, the item can be eliminated, or has a low priority . As can be observed, the tasks deal with topics, such as high - risk pregnancy, breast feeding, and family planning . The remaining tasks (45 tasks) are the needs; we should focus on meeting them . With regard to the results obtained and considering the viewpoints of the needs assessment team, 22, 18, 26, 38, 43, and 44 to the major, and as the needs assessment team agreed, these items were not eliminated, but received lower priority in educational planning . We took advantage from expert s opinions in evaluating the tasks of msc graduates of reproductive health . In this respect, the tasks extracted were consistent with the framework of reproductive health provided by the who and the american guideline of educational planning . However, according to the differences in problems of our country with those of the others, the list of tasks was not exactly similar to any list prepared for other countries . Altschuld method was used to perform the priority- setting in needs assessment, because the major did not exist before . Thus, it was not possible to determine the gap between the present and desirable states . Moreover, there are currently other specialties that provide services in the same field in the health system . So, in needs assessment, the importance of tasks defined for meeting the needs of people s reproductive health were considered, as well as the unavailability and feasibility of the services . The results showed that in experts opinion, all the tasks extracted were important for the graduates, and most of the tasks, 46 out of 57, were not currently provided . These tasks were related to management and planning, education and consultation about the puberty and pre- puberty health and the issues related to them, sexual health, elderly health, menopause, empowerment of women, and participation of men . The experts believed that the items were important (all scored above 4), and were not available for the people . According to the experts, of the tasks proposed 10 items these items were feasible; however, since they were available and were provided by graduates of other specialties, the items were not given high priorities . The needs that do not require further action according to the priority - setting scale . Thus, it is not necessary to train new specialists to provide these services . With regard to the remaining tasks, the experts believed that the services were not provided at all or provided to degrees less than somehow . Moreover, except for management and planning for changing of gender beliefs and superstitions of families and the society, on which the experts did not agree, all other items were feasible . Therefore, these services should necessarily be provided by some individuals or systems . In this regard, general evaluation of the tasks extracted indicated that the process of defining tasks of reproductive health was performed multi dimensionally and accurately . The tasks defined cover all stages of reproductive age and all feasible duties in the field of health . Reproductive health for the two ends of reproduction age, that is, adolescence and higher ages, are important issues in iranian reproductive health . This is because although almost 34% of the current iranian population is young, the elderly population in urban areas has been grown fourfold in the past 30 years . Furthermore, in near future, a high percentage of the population would be old, and consequently the reproductive health needs associated with these ages would become important . Management and planning, consultation, education and other items related to reproductive health in older age groups of men and menopause of women were of the main tasks defined for the major . These tasks can be of the current and future tasks, which should receive appropriate attention . This is due to the increasing growth in elderly population in iran, as it is estimated that in 2031, elderly population boom will occur in iran, and 25%-30% of the population will then be above 50 years of age . The tasks related to sexual health were among the tasks that were considered in all stages of life . In spite of obtaining high scores, the tasks of education and consultation about the gender and sexual roles were defined in this regard . Many studies have been carried out on adolescents, and showed that the adolescents were unfamiliar with sexual issues, and there is a need for education and consultation of adolescents in this respect. [1821] some researchers have evaluated gender roles of men and women and found the problems of this field . They recommend that by pre and post marriage consultation, the couples should become familiar with mental schema and cultural clich, and the influence of these factors on mental assessment of sexual arousal and satisfaction with sexual relationship . Moreover, the importance of these factors should be taught to decrease the problems of married life . Considering the tasks defined for the major, the graduates can more specifically meet the needs of the society in this respect . A branch of this item is violence against women, which is a topic proposed by the who as an item for assessment and follow up in reproductive health . Some researchers believe that violence against women and children within the families has decreased; however, the problems require education with the aim of changing the behavior . Department of reproductive health in the who with the aid of the united nations population fund (unfpa) and human reproduction program (hrp) has defined the main topics of reproductive health . These topics would be helpful in research on reproductive health and making the policy makers, scientists, health care providers, physicians, consumers, and representatives of the society aware of the research priorities in development of reproductive and gender health . The topics were namely, older age and reproductive and gender health, family planning, women s circumcision, and other harmful practices, infertility, mothers health and abortion, urinary tract infections, sexually transmitted diseases, aids, relationship between reproductive health and aids, unsafe abortions, and violence against women . Considering the health state of our society and frequency of the problems, however, some less frequent topics in our country, such as women s circumcision, were not placed among the main topics, and considering the young population of iran, some topics such as those related to adolescents were mentioned as the main topics in reproductive health needs . Some limitations of the study were obligation in selection of the participants from the specialists of the field, because they were mostly busy and did not have enough time to answer the questions . To overcome this problem, the study was better to be performed in a larger time interval and during academic off day . Considering the results obtained, the authorities should develop the reproductive health major in subgroups, such as sexual health, research in reproductive health, and reproduction rights, and also modify the curriculum of courses related to the field for students of medical sciences . Furthermore, researchers should carry out more studies on the neglected tasks of physicians in reproductive health.
Continuous care delivery demands that health care providers lead patients smoothly through a chain of care that includes a number of professionals, departments and organizations, each of which are necessary for the provision of quality care at the right time and in the right place for each and every patient [15]. Experience has taught us that this process is complex . Indeed, in countries like sweden, england, the netherlands and austria, hospitals struggle with a bed - blocking problem . A bed - blocker is a patient who has completed treatment in one part of the care chain (e.g. A hospital) and is waiting for admittance to the next part of the chain (e.g. A nursing home or home care)., it impacts health care costs as an occupied bed in a hospital is more expensive than an occupied bed in a nursing home or, alternatively, an occupied bed at home . Hospitals have attempted to tackle the bed - blocking problem by setting up departments or areas that handle bed - blockers . In austria, these are called after care areas . In the netherlands, where in 2006 6.1% of all hospital days were bed - blocking days an icd is a nursing department usually situated in the hospital building that functions as a buffer when the hospital is over its capacity . Initially, the establishment of icds appeared to be a promising and effective solution to bed - blocking . In fact, between 1999 and 2002, the total number of bed blocking days in hospitals with an icd decreased by 30% while hospitals without an icd saw a decrease of only 15% . However, with the establishment of icds, another problem arose, namely queues for admission to the icds . Although the average waiting time and the size of the queue had decreased between 2003 and 2006 from 11 to 8 days and from 8 to 7 patients, respectively, the queue remained [7, 9]. In essence, the solution (i.e. Icds) generated the same problem it was meant to solve (i.e. Waiting times and bed - blocking). Previously, we explained that this was likely caused by a lack of buffer management (bm), a tool that endeavors to balance patient flow in the hospital to nursing home chain of care . We hypothesized that bm was not applied by health care professionals and management because of a number of negative beliefs regarding bm . We further concluded that bm is indeed the most promising way for increasing the effectiveness of buffering . However, we must question whether our assumption that providers and managers hold negative beliefs about bm is correct . We are also interested in what can be done to make bm more feasible in health care settings . With this in mind, this paper seeks to firstly explain the theory of bm and then discuss the tenability of our previous assumptions given the results of (explorative) research recently conducted . Bm builds upon the theory of constraints drum - buffer - rope [1117]. It aims to maximize the throughput in the chain by firstly identifying impeding factors or constraints and then taking measures to manage them properly . Constraints can be identified by analyzing the sub - processes of the (patient) flow in the chain . The most impeding factor is called the system s bottleneck . Because all sub - processes are linked to one other, the bottleneck determines the flow of the entire process and thus requires immediate and appropriate attention . In identifying the bottleneck, when the bottleneck is found, the drum - buffer - rope method is used to balance the patient flow in the whole chain . In the dutch hospital to nursing home or home care chain of care, the patient flow into the nursing home is the bottleneck [7, 9]. The drum - buffer - rope method prescribes determining how many patients should be admitted to the hospital as well as the throughput from the hospital to the nursing home via the icd using a so - called rope . When the buffer overflows, the rope, which is an information stream, closes the hospital doors for potential (elective) buffer patients, which are mainly patients with mobility problems, rehabilitation needs or cardiovascular problems [7, 9]. This procedure is illustrated in figure 1, which displays three situations: the chain with no buffer (situation a), the chain with a buffer but without bm (situation b) and the chain with a buffer and bm (situation c). Correct execution of bm requires that the following prerequisites for successful bm be taken into account [13, 18]: in order to balance the flow, the entire process including its sub - processes must be considered.total flow can only be effectively increased by increasing the flow in the bottleneck.in order to prevent queues, the buffer needs free space to absorb fluctuations which are most frequently caused by non - elective patients . A maximum occupancy of 70% is considered acceptable.the use of a rope to adjust the input rate at the beginning of the process is necessary to prevent overflow in the sub - processes . In order to balance the flow total flow can only be effectively increased by increasing the flow in the bottleneck . In order to prevent queues, the buffer needs free space to absorb fluctuations which are most frequently caused by non - elective patients . The use of a rope to adjust the input rate at the beginning of the process is necessary to prevent overflow in the sub - processes . In practice the abovementioned prerequisites for effective bm are infrequently considered and the rope is rarely used . Another misconception is that if the sub - processes are optimized, the flow of the entire process will increase . In fact, this leads to an unbalanced flow because the flow is determined by the bottleneck . With respect to bed - blocking, what will happen is the following: because patient flow in nursing homes is lower than patient flow in hospitals and because fluctuations at the entrance cannot be absorbed, in the absence of bm, the icd will overflow . As mentioned earlier, a queue will form . This will result in bed blockers in the hospital (bb) who are waiting to be transferred to the icd . Although this appears to be pretty straightforward, the problem of bed - blocking nonetheless remains . It is thus important to better understand why care providers involved fail to fulfill the abovementioned prerequisites . One possible explanation is that management and professionals in hospitals and/or nursing homes hold one or more negative beliefs about bm . We have previously provided objections to these beliefs . The beliefs and our objections are: buffer management is unnecessary.the argument is that bm is unnecessary when discharge management is a sufficient and more manageable alternative [cf . However, discharge management does not balance patient flow nor does it establish a figurative tap to prevent additional patients per time unit from entering the chain when upstream capacity is limited . Although discharge management, when used effectively, is a helpful coordination tool, research has indicated that discharge management has many problems with respect to, among other things, communication and information exchange .patient arrivals are unpredictable.the argument is that fluctuations in patient flow with respect to the frequency and intensity of patient arrivals cannot be predicted . Patient flow is predictable when elective and non - elective admissions are distinguished from one another [cf . Additionally, although it might not be possible to predict the exact number of emergent arrivals per day, the size of emergent demand generally appears to be quite stable and predictable over time . As such, we contend that the required size of the departments and buffer can be defined.buffer management is unethical.the argument is that putting a figurative tap on patient flows is ethically unacceptable as it implies the refusal of some patients [cf . We ask whether it is safe and ethically acceptable to treat (elective) patients without knowing if they can be nursed and cared for afterwards [cf . Apparently, it is impossible to provide accessible high quality care that is tailored to the needs of the patient . We contend that we must be aware of this dilemma and subsequently take conscious clinical and political decisions.care organizations are unwilling to cooperate.cooperation between care providers using a chain perspective is necessary in order to maintain the delicate balance in the chain . Previous research has indicated that care organizations are often unwilling to cooperate [5, 32] thus supporting this argument against bm . However, we contend that the current trend in many european countries actually demonstrates an increased awareness of the importance of cooperation among most care providers [5, 33, 34]. The argument is that bm is unnecessary when discharge management is a sufficient and more manageable alternative [cf . . However, discharge management does not balance patient flow nor does it establish a figurative tap to prevent additional patients per time unit from entering the chain when upstream capacity is limited . Although discharge management, when used effectively, is a helpful coordination tool, research has indicated that discharge management has many problems with respect to, among other things, communication and information exchange . The argument is that fluctuations in patient flow with respect to the frequency and intensity of patient arrivals cannot be predicted . Patient flow is predictable when elective and non - elective admissions are distinguished from one another [cf . Although it might not be possible to predict the exact number of emergent arrivals per day, the size of emergent demand generally appears to be quite stable and predictable over time . As such, we contend that the required size of the departments and buffer can be defined . The argument is that putting a figurative tap on patient flows is ethically unacceptable as it implies the refusal of some patients [cf . We ask whether it is safe and ethically acceptable to treat (elective) patients without knowing if they can be nursed and cared for afterwards [cf . Apparently, it is impossible to provide accessible high quality care that is tailored to the needs of the patient . We contend that we must be aware of this dilemma and subsequently take conscious clinical and political decisions . Cooperation between care providers using a chain perspective is necessary in order to maintain the delicate balance in the chain . Previous research has indicated that care organizations are often unwilling to cooperate [5, 32] thus supporting this argument against bm . However, we contend that the current trend in many european countries actually demonstrates an increased awareness of the importance of cooperation among most care providers [5, 33, 34]. Van hartingsveldt conducted three small case studies to further explore care providers beliefs about bm (regarding both the sub - processes and the chain as a whole) and to investigate whether or not providers held the four negative beliefs about bm as we assumed . The case studies were derived from qualitative data gathered in a larger mixed methods research project, using questionnaires and interviews with top - managers that addressed the availability of icds in all dutch hospitals and their functioning in terms of patient flows, waiting times, manpower, task division and responsibilities . The data gathered in 2006 were compared with data from a similar study conducted in 2003 in order to determine if things had changed over the years . One case study was conducted with an icd that had an average outcome, another with an icd that had a lower than average outcome and yet another with an icd that had a higher than average outcome . In each case study, an in - depth interview was conducted with icd executives using a structured interview protocol . The managers of the selected icds were interviewed and asked to mention two additional interview candidates, one that is responsible for referring patients to the icd and one that is responsible for admitting patients to nursing homes . Consequently, in each case study, three people with different work positions were interviewed . In total, nine interviews were conducted . Hermeneutic and narrative approaches were applied in the analyses . Based on the assumption that respondents answers derive meaning from the whole and the whole derives meaning from the parts, the interviews were read and reread in an effort to develop a comprehensive understanding of the data . The interview data showed the following: all respondents considered the hospital, the icd and the nursing home to be separate links in the care chain . In their view, the organizations are independent . Meetings that aimed to enable optimal patient flow and that included representatives of the three organizations failed to discuss whether equal numbers of patients were being admitted and discharged from the icd.because icds are financed according to how many beds are occupied, it is not surprising that icd respondents emphasized striving for a 100% occupancy rate in order to gain sufficient funding . This was also illustrated by the following citations: we celebrated the moment that our department was fully occupied; currently we have an occupancy rate of 70% . I am therefore searching for patients to stay in the icd so that i can reach my production target.all icd and nursing home respondents said that they did not have any information regarding planned patient referrals from nursing departments and icds, respectively . This was confirmed by respondents from nursing departments referring to icds and respondents from icds referring to nursing homes.one nursing home respondent said that she did not benefit from investing energy into the facilitation of patient flow from the icd to the nursing home as the number of patients being admitted to nursing homes is already high enough to fill all the nursing home beds.all respondents shared objections to the contention that bm is unnecessary . More specifically, they indicated that insurers do not reimburse empty beds.all respondents did not agree with the contention that patient flows are unpredictable and that this serves as an inhibitor to bm . They, in fact, claimed that health care providers always have to deal with unpredictability.none of the respondents felt that bm is unethical . They indicated that patient refusal is simply part of daily practice in health care . According to one icd respondent: also here at the icd . Then you have to refuse patients . That s just how it is in practice.all respondents indicated that cooperation is necessary in order to enable optimal patient flow and in order to reduce the number of bed - blockers . All respondents considered the hospital, the icd and the nursing home to be separate links in the care chain . In their view, the organizations are independent . Meetings that aimed to enable optimal patient flow and that included representatives of the three organizations failed to discuss whether equal numbers of patients were being admitted and discharged from the icd . Because icds are financed according to how many beds are occupied, it is not surprising that icd respondents emphasized striving for a 100% occupancy rate in order to gain sufficient funding . This was also illustrated by the following citations: we celebrated the moment that our department was fully occupied; currently we have an occupancy rate of 70% . I am therefore searching for patients to stay in the icd so that i can reach my production target . All icd and nursing home respondents said that they did not have any information regarding planned patient referrals from nursing departments and icds, respectively . This was confirmed by respondents from nursing departments referring to icds and respondents from icds referring to nursing homes . One nursing home respondent said that she did not benefit from investing energy into the facilitation of patient flow from the icd to the nursing home as the number of patients being admitted to nursing homes is already high enough to fill all the nursing home beds . All respondents did not agree with the contention that patient flows are unpredictable and that this serves as an inhibitor to bm . They, in fact, claimed that health care providers always have to deal with unpredictability . They indicated that patient refusal is simply part of daily practice in health care . According to one icd respondent: also here at the icd, we are sometimes fully occupied . . Then you have to refuse patients . That s just how it is in practice . All respondents indicated that cooperation is necessary in order to enable optimal patient flow and in order to reduce the number of bed - blockers . At first sight, the interview data suggest that our previous assumption that health care providers and management hold negative beliefs about bm is incorrect . For, they broadly supported the necessity and feasibility of bm [cf . 57]. In practice, however, they do not apply the bm solution and they do not cooperate as is needed for bm application . For, although respondents said that cooperation is most valuable and necessary [cf . They failed to share information with one another; they failed to provide feedback about patient flows to one another; and they sought to meet the interests of their own department or organization (e.g. Acquiring maximum funding) before considering the interests of the total chain and its patient flow [cf . They quite simply failed to cooperate in ways that are imperative to the success of bm . Such a paradox between words and actions has previously been referred to as skilled incompetence by argyris . In order to shed light on why, when it comes to cooperation, respondents actions did not coincide with their words, we will further explore the inhibiting and promoting conditions for cooperation below . In seeking to understand cooperation between organizations, organizational units or professionals in health care, we have opted to borrow from organizational theories and literature on integrated care . Organizational theories claim that organizational operations, like cooperation, communication, governance and knowledge transfer, are, in the end, determined by actions . Actions are, in turn, dependent on the willingness and ability of those involved [3639]. One of the basic conditions for willingness to cooperate is the presence of interdependency between the organizations involved [3941]. In health care, interdependency among providers is rapidly increasing as pressure to coordinate services and deliver integrated care is increasing [2, 4, 42, 43]. A second basic condition for willingness to cooperate is the presence of at least one common goal [5, 39, 44]. In health care, common goals are often construed in one s mind but not manifested in one s actions . A third basic condition is that the cooperation contributes to the department or organization s own goals [4447]. With the resource dependency theory in mind, we assume that interdependency increases when the number and weight of common goals and the benefits of cooperation for the organization s involved increases . We also assume that greater willingness generates more opportunities to act . In order to act means include money, personnel, time, equipment and accommodation . In health care,, cooperation always requires transaction costs in order to generate revenues later . This combination of scarce resources and transaction costs for cooperation does not promote providers abilities to cooperate . Furthermore, limited ability is likely to reduce willingness . In most health care systems, the availability of means is dependent on legal and financial structures and regulations [3, 34, 49]. Most countries have some laws and regulations fostering cooperation and others that hinder cooperation [5, 33, 49]. The availability of means is also dependent on managerial capabilities [38, 50], especially political and negotiating skills but also certain leadership characteristics, such as transformational leadership (vision, ability to motivate and convince) and intrinsic personal leadership features, such as charisma, charm and attractiveness [38, 5053]. Our previous research on integrated care revealed that managerial capabilities, especially leadership features and behavior, were strongly related to success or failure of integrated care networks [44, 46, 47]. Cooperation is not simply determined by promoting and inhibiting conditions . In the real world, the willingness and ability to act in cooperative ways is also dependent on the answers to two questions . The first is, how do those involved perceive the abovementioned conditions? [cf . According to the thomas theoremif men perceive things as real, they are real in their consequences the perception of the situation affects the actions taken . The second question concerns the extent they are able to change the routines, principles and beliefs (rpbs) employed in their daily work in order to achieve successful cooperation . We must ask if they are able to shift their focus from their own organization or department to the chain as a whole . Are they able to find solutions for financial problems and develop trust in their partners instead of harboring distrust and prejudice? Are they able to replace their belief in their own power with a belief in shared power? It is likely that the ability to change rpbs is in part dependent on perceptions while rpbs also impact perceptions . As such, perceptions and this is a vicious cycle and experience has taught us that breaking vicious cycles is often a laborious endeavor . One of the basic conditions for willingness to cooperate is the presence of interdependency between the organizations involved [3941]. In health care, interdependency among providers is rapidly increasing as pressure to coordinate services and deliver integrated care is increasing [2, 4, 42, 43]. A second basic condition for willingness to cooperate is the presence of at least one common goal [5, 39, 44]. In health care, common goals are often construed in one s mind but not manifested in one s actions . A third basic condition is that the cooperation contributes to the department or organization s own goals [4447]. With the resource dependency theory in mind, we assume that interdependency increases when the number and weight of common goals and the benefits of cooperation for the organization s involved increases . Means include money, personnel, time, equipment and accommodation . In health care, means are always scarce . To make matters worse, cooperation always requires transaction costs in order to generate revenues later . This combination of scarce resources and transaction costs for cooperation does not promote providers abilities to cooperate . Furthermore, limited ability is likely to reduce willingness . In most health care systems, the availability of means is dependent on legal and financial structures and regulations [3, 34, 49]. Most countries have some laws and regulations fostering cooperation and others that hinder cooperation [5, 33, 49]. The availability of means is also dependent on managerial capabilities [38, 50], especially political and negotiating skills but also certain leadership characteristics, such as transformational leadership (vision, ability to motivate and convince) and intrinsic personal leadership features, such as charisma, charm and attractiveness [38, 5053]. Our previous research on integrated care revealed that managerial capabilities, especially leadership features and behavior, were strongly related to success or failure of integrated care networks [44, 46, 47]. Cooperation is not simply determined by promoting and inhibiting conditions . In the real world, the willingness and ability to act in cooperative ways is also dependent on the answers to two questions . The first is, how do those involved perceive the abovementioned conditions? [cf . According to the thomas theoremif men perceive things as real, they are real in their consequences the perception of the situation affects the actions taken . The second question concerns the extent they are able to change the routines, principles and beliefs (rpbs) employed in their daily work in order to achieve successful cooperation . We must ask if they are able to shift their focus from their own organization or department to the chain as a whole . Are they able to find solutions for financial problems and develop trust in their partners instead of harboring distrust and prejudice? Are they able to replace their belief in their own power with a belief in shared power? It is likely that the ability to change rpbs is in part dependent on perceptions while rpbs also impact perceptions . This is a vicious cycle and experience has taught us that breaking vicious cycles is often a laborious endeavor . With respect to the icd case, we must question whether the dutch hospitals and nursing homes are aware of their interdependency on a daily basis . It is quite likely that hospital staff members (doctors, interns, administrative personnel) are unaware of their dependency on nursing home capacities when admitting potential buffer patients . At the same time, nursing home staff members probably do not realize that their hampered patient flow causes waiting lists for hospitals . Furthermore, we must question whether the hospitals and nursing homes are truly aware of a common goal . Most hospitals and nursing homes do formulate common goals to improve patient flow in a formal agreement [7, 9] but are they really making decisions on the basis of these goals in their daily work? It is likely that some parties in some parts of the chain do not even feel that improvement of the patient flow is necessary . This seems to be the case in nursing homes where the patient flow into the nursing home is sufficient to fill the available beds . We must also question whether hospitals and nursing homes are really aware of bm and how it can contribute to the attainment of an organization or department s own goals . Instead, they most often consider bm to be a threat to their own goals . They may believe that bm threatens their autonomy and the acquisition of sufficient funding . At the same time, they fail to tackle the impeding factors that make bm a threat . For instance, with respect to funding, they often do not realize that it may be worthwhile fighting the prevailing system for the provision of funding . It may also be worthwhile to seek other resources or work more efficiently in order to enlarge their ability to cooperate . As such, it appears that the advantages of the icd buffer and bm; namely improved patient flow and increased nursing home capacity due to additional beds in hospitals, is not perceived, or is at the very least, not perceived as important enough to change the rpbs of those involved . Our conclusion is that, in health care practice, three of the four negative beliefs regarding bm can be refuted . Although the providers involved in the case studies perceived a need for cooperation, they were not able or willing to translate this into action . In addition, a shortage of means caused by inappropriate incentives from financiers hampered concrete efforts to improve cooperation . In fact, the financial structure may very well be used as an argument against cooperation . We must realize that the current behavior is difficult to change as it is part of a vicious cycle of rpbs and perceptions, both of which are not in favor of cooperation . More importantly, a major reason for not favoring cooperation has institutional roots [cf . 55], more specifically, the health care system s financial structures, rules and regulations as well as its professional values (i.e. Professional autonomy). In essence, providers skilled incompetence with respect to cooperation is in part an institutionalized skilled incompetence that is very difficult to tackle . In order to make bm not only theoretically feasible, but also practically applicable, essentially, it boils down to a shift of focus from parts (i.e. Each individual organization) to the whole (i.e. The flow between participating organizations). Only then theoretical acceptance of bm will be followed by consistent bm actions . For this to be achieved, we recommend the promotion of providers willingness and ability to take actions which foster cooperation (i.e. The heart of our cooperation model figure 2). Two (change) strategies could be followed simultaneously: first, a negotiating strategy, to create win - win - situations, and second, a learning strategy, on how to adopt cooperation in the daily routines, anchored in people principles and beliefs [cf . Several interventions at the system s macro, meso or micro level are possible . Examples of interventions related to the negotiating strategy are: convincing politicians and other powerful stakeholders to change the system such that incentives for cooperation are present as this is a macro - level intervention, it is an important job for top managers, but also for occupational organizations . Making care providers and professionals aware of the chain perspective . Convincing them that adopting this perspective is needed to solve their bed - blocking problems and, by doing that, improve quality and save costs . More concrete: an icd manager could stress the chain perspective and its advantages on the meso and micro levels, in discussions with medical professionals, hospital governors and nursing home managers . Negotiating with care insurers or health authorities in order to enlarge financial means for cooperation . It is important to stress cost savings on the macro level, related to successful cooperation . Striving for funding based on cooperation between organizations (i.e. Flow funding) instead of funding the participating organizations individually (as is the case now). Appointing a chain manager or director that can oversee the entire care process and consider the prerequisites for bm in this process . Appointment of a chain manager should be a common action on meso / micro level, i.e. The (top) managers of the organizations involved in the chain, with commitment of professionals . The chain director should be provided with the right competences, like transformational leadership and have appropriate negotiating skills as this can promote change in providers rpbs . Drafting and signing agreements that ensure provider compliance with the bm prerequisites, including obligations and potentially even sanctions . The chain manager (at meso level) could prepare this in cooperation with the organizational managers and professionals involved . Examples of interventions related to the learning strategy are: learning to find and use other (local) sources at macro and meso levels to finance a care process that allows for the regulation of hospital intakes based on icd bed occupancy . This learning can be stimulated in brainstorm sessions of professionals and providers involved (meso and micro level). Motivating and teaching professionals and providers involved to develop common goals and to come to common adherence based on those common goals . Higher management and the chain manager could take initiatives in this direction by first mobilizing professionals who understand the chain perspective and are motivated to convince their colleagues of the advantages of cooperation . Setting up a virtual network organization to manage cooperation and learn how to make use of the virtual organization (meso level). Ict is a helpful tool to execute cooperative actions, but it takes time to become familiar with using it . Use of ict experts can be helpful, but more important is that ict becomes part of providers daily routines . Table 1 gives an overview of the two strategies, and the related interventions on macro, meso and micro level of the health and social system . It depends of the individual situation what interventions to choose and when to apply them . In addition, we realize that the chain manager s task is not a simple one . Breaking down is that the chain manager is or makes him / herself accepted by those involved . If not, it will be difficult to realize the conditions for cooperation, as mentioned in figure 2 . So, the interventions require investments, and the return on investment in terms of quality and savings should be analyzed in advance . Our conclusion is that, in health care, the failure to cooperate is a serious inhibitor of bm . Continuous persistent and purposeful efforts to improve cooperation between care providers are therefore imperative, as people often relapse into old routines, principles and beliefs . Maren sogstad, associate professor, phd, centre for care research, gjvik university college, post box 191, 2802 gjvik, norway birthe dinesen, phd, associate professor, department of health science and technology, aalborg university, fredrik bajersvej 7 d1, dk-9220 aalborg, denmark jason ch yap, mbbs, mmed (public health), fams, mba(is), chief (health information & innovation), agency for integrated care, singapore
This review focuses on advances in analytical technologies for high - throughput (htp) glycomics (i.e. The large - scale study of glycoconjugate glycosylation patterns). Our emphasis will be on glycomics to support (1) realization of glycoprotein biopharmaceuticals and (2) development of glycan biomarkers of disease for clinical diagnostics . Glycans play key roles in important biological processes such as protein folding, host - pathogen interaction and signal transduction . They are present both in free form, or bound to proteins or lipids, and can modify the biological activities of the conjugate . The glycomics technologies covered in this review are mainly for analysis of protein glycosylation, the major types of which are n - linked glycans attached at asn - x - ser / thr motifs on the peptide backbone and o - linked glycans attached to ser / thr . Protein glycosylation is a co - translational and post - translational modification where glycans are attached to proteins during translation, assisting in the folding and quality control of the protein . Attached glycans are subsequently modified in the endoplasmic reticulum and golgi to varying levels of complexity, causing glycosylation to be one of the most complex and diverse types of protein modification . Glycomics is of interest to biopharma companies because glycans can greatly modify safety and efficacy profiles of the therapeutic protein to which they are attached . For example, igg effector functions such as antibody - dependent cellular cytotoxicity and complement - dependent cytotoxicity of monoclonal antibodies are modulated by n - glycosylation of its fragment crystallizable (fc) portion . N - glycosylation modifications are also involved in determining the plasma half - life of glycoproteins by modulating the interaction with various receptors [4, 5]. Even small changes in glycosylation can lead to serious issues such as anaphylaxis in patients and the destruction of therapeutic activity . These effects have been found in drugs such as cetuximab (a monoclonal antibody (mab) for treatment of metastatic colorectal cancer), herceptin (a mab for treatment of breast cancer) and erythropoietin (epo a drug for increasing red blood cells in patients with renal failure and cancer patients undergoing chemotherapy). Given this, there are now increasing pressures from regulatory authorities for drug manufacturers to measure, optimize and control their drug s glycosylation . This task is very challenging given the structural complexity of biopharmaceutical glycosylation and achieving it has required adoption of a new approach to drug design namely quality by design (qbd). This methodology has been actively promoted by the fda and other drug regulators and is being adopted by leading biopharma companies as the system of best practice for the design, development, and manufacture of biologic drugs . Qbd has the potential to deal with the complexities of drug glycans and could help simplify difficult tasks such as demonstrating comparability of biopharmaceutical glycosylation . Studies of the qbd approach by the cmc biotech working group (a consortium of experts from leading biopharma organizations including genentech, amgen, eli lilly, and pfizer), indicate that definition of the qbd design space may require analysis of tens of thousands of drug samples . Successful adoption of qbd for development of glycoprotein therapeutics requires glycoprofiling systems that can cope with this very high level of sample throughput in effect it will need to be built on a glycomics framework . In addition to its use in biopharmaceutical realization, there is also increasing interest in glycomics for development of new clinical diagnostics based on glycosylation markers of diseases . This follows from the key roles that glycans play in major biological processes from the fertilization of eggs by sperm to cell death and their involvement in a multitude of disease etiologies and progressions . The potential of glycans as sensitive disease biomarkers is particularly high, since they are synthesized by the concerted action of multiple proteins, and influenced by various disease - associated factors such as genetic variations, epigenetic signatures and metabolic distortions . Glycan patterns of secreted proteins often confer information on the pathophysiological status of the secreting cells, making glycosylation analysis of relevant glycoproteins a suitable diagnostic tool . Changes in glycosylation are associated with various diseases like cancer, neurodegenerative and inflammatory diseases [915]. These types of associated changes have resulted in an increased interest in studying the alterations in glycosylation patterns of biological fluids as disease biomarkers as well as for patient stratification and personalized medicine . To date, two glycan biomarkers have been introduced for routine clinical diagnostics, namely carbohydrate - deficient transferrin for the detection of alcohol abuse and fucosylated serum alpha - fetoprotein for the early diagnosis of hepatocellular carcinoma . Importantly, glycomic changes are not limited to single conditions but are rather a hallmark of virtually any human disease, as stated recently in a whitepaper by walt et al . And diverse promising glycomic biomarkers have been described for mody - type diabetes and immunoglobulin g myeloma [19, 20]. However, most of the glycan biomarkers discovered so far have been studied using glycoanalytical technologies that would not be suitable for use in routine clinical diagnostic labs the shortfall being due mainly to limitations in sample throughput, resolution and affordability of the methods . Given the above, it is clear that development of reliable, affordable, high - resolution htp glycomics technologies would be essential in the biopharma industry for efficient optimization of the glycosylation patterns of glycoprotein therapeutics as well as advancement of practical clinical diagnostics based on glycosylation biomarkers of disease . The rest of this review will cover a number of recent advances in glycomics technologies for these two applications . It highlights current challenges in analytical glycomics methods and presents some approaches of how the bottlenecks in protein glycosylation analysis may be overcome . Many reviews describe glycan sample preparation steps such as n- and o - glycan release, de - glycosylated sample desalting and clean - up, derivatization of released glycans and analysis using different analytical platforms [2125]. Here next to their application in biomedical research, these methods are often applied for the characterization of biopharmaceuticals . Liquid chromatography (lc) is a widely used glycoanalytical technique with good quantification, reproducibility, and separation of glycan isomers . Have demonstrated a htp sample preparation for high performance lc (hplc) using a polyvinylidene fluoride (pvdf) membrane and an in - gel block method for glycan release . The enzymatic release step is followed by 2-aminobenzamide (2-ab) labeling and purification using microplates, and subsequent hplc analysis . Automation of the procedure was achieved to a large extent, as well as the automated analysis of the obtained results, but the resulting method is still labor intensive, particularly the in - gel block preparation and glycan release stage . Performing the whole process on 96 samples takes 3 days with an additional 2 days for the acquisition of the chromatographic data . Used the rapid deglycosylation kit and instant 2-ab kit from prozyme to demonstrate an approach that only took 3.5 h of sample preparation time . A downside here is that the required kits are a considerable cost factor, which may limit the number of samples that can be studied . Burnina et al ., on the other hand, have described a cost effective htp sample preparation method for the characterization of therapeutic antibody n - glycosylation taking approximately 90 min . The workflow included the following steps, denaturation, reduction, deglycosylation using a hydrophobic immobilon - p pvdf membrane filter plate, fluorescent labeling and clean - up using a 96-well hydrophilic filter plate . They demonstrated the use of orthogonal assays and obtained lc and ms which were in agreement . [2931] reported capillary gel electrophoresis with laser - induced fluorescence detection (cge - lif) using a dna sequencer for the analysis and profiling of total serum n - glycans labeled with the negatively charged, fluorescent label 8-aminopyrene-1,3,6-trisulfonic acid (apts). Have demonstrated the htp application of this by optimizing sample preparation and employing a system with multiplexed capillaries . After protein denaturation and n - glycan release, the glycans were labeled with apts in a 96 well plate and subjected to hydrophilic interaction chromatography (hilic) purification . Using the multiplexed cge - lif system, 48 samples can be analyzed in parallel (scalable to 96 samples) allowing the analysis of 96 samples with a hands - on time of 2.5 h. a recent large - scale application making use of the multiplexed cge - lif system is the analysis of alpha1-antitrypsin and immunoglobulin a from over 2,400 human plasma samples . Set up to discover novel biomarkers, the method revealed protein glycosylation to be associated with age and sex, as well as with cardiovascular and metabolic diseases . Varadi et al . Described the use of magnetic beads for rapid, htp sample preparation of n - glycans from therapeutic antibodies and indicate that this method is easily automatable . The carboxyl coated magnetic beads are capable of capturing released n - glycans due to the ionic interaction between the positively charged glycosylamines and the negatively charged carboxylated beads . The glycans were captured using the magnetic beads and were labeled with apts for 2 h. once again the magnetic beads were used for binding the labeled glycans and elution was accomplished by washing the beads with water . Mass spectrometric analysis of glycans is difficult when dealing with isobaric structures and particularly difficult when dealing with sialylated species . Sialic acids are known to degrade under harsh sample preparation and ionization conditions, as well as showing a strong preference towards negative ionization . Sialylated glycans are often derivatized before mass spectrometric analysis, either by carboxylic acid - specific methods or permethylation . Gil et al . Reported a one pot sialic acid amidation reaction using acetohydrazide, allowing neutralization of sialylated n - glycans still attached to proteins . Accidental schiff base formation with the acetohydrazide is prevented because the glycans are still attached to the protein . N - glycan release could then be performed afterwards, as well as labeling with the negatively charged fluorescent label 2-aminobenzoic acid (2-aa). Clean - up was performed by 96-well filter plate, and analysis by maldi - time of flight (tof)-ms . The authors used recombinant human glycoproteins to determine changes in fucosylation and changes in sialylation and compared the maldi tof ms data with hilic hplc data proving that they were in good agreement . Another approach used by jeong et al . For htp quantitative n - glycan analysis was solid phase permethylation . Sialylated n - glycans were protected with a methyl group at the carboxylic acid by the permethylation reaction, and glycan mixtures were subsequently analyzed by maldi sample preparation steps were performed in a 96-well format, comprising glycoprotein binding on pvdf, deglycosylation, purification of n - glycans by porous graphitized carbon (pgc), and solid phase permethylation . By spiking a known concentration of internal standard mixture with ovalbumin and porcine thyroglobulin, the authors obtained absolute quantitation for these samples, as well as relative quantification of 49 glycans from human serum prostate specific antigen . The complete sample preparation utilizing this method takes 2 days, but is quite labor intensive due to multiple manual pipetting steps . Miura et al . Have developed a protocol for the methyl esterification of sialic acids using 3-methyl-1-p - tolyltriazene (mtt) as sole reactant and methyl group donor . This derivatization was performed on both the free n - glycans, requiring a subsequent purification step, as well as after immobilization of the n - glycans on affi - gel beads, allowing a much increased throughput . Ms with significantly increased stability . Using this method, both the neutral glycans and the previously negatively charged sialylated glycans could be observed in the same positive mode spectrum . Liu et al ., have shown a similar increase in sialic acid stability after methylamidation of the carboxylic acid residues with a combination of methylamine and tripyrrolidinophosphonium hexafluorophosphate (pyaop). They have shown this approach to be suitable for the derivatization of o - acetylated sialic acids, and were able to perform both maldi and electrospray ionization (esi)-ms (/ms) experiments on the glycans . Both the mtt and the pyaop stabilization methods only require a few hours of preparation time (purification included), making them suitable for htp glycomics . Another variant of methods for sialic acid modification can make use of the chemical nature of 2,3-linked sialic acids to lactonize with the subterminal galactose resulting in a loss of water that can be observed in mass spectrometry, whilst 2,6-linked sialic acids show no such behavior . Published by wheeler et al ., the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (dmt mm) in combination with methanol showed good reaction selectivity for the sialic acid linkages, generating either lactones or methyl esters . Alley et al . Have used dmt - mm to generate amides instead of esters, performing the reaction in the presence of ammonium chloride . Subsequent permethylation still showed linkage - specific mass differences, with the added benefit of more informative fragmentation spectra . Another extension of the dmt - mm deriviatization method was shown by tousi et al . Who performed nano - hilic - orbitrap - ms of the modified glycans, effectively increasing the number of individual resolvable peaks after hilic separation, as well as allowing linkage - specific ms(/ms). Similar to mtt and pyaop, dmt - mm - assisted modification can be performed in less than 3 h (with the reaction conditions and reaction time depending on the modification to be introduced), and is highly suitable for htp sample preparation . An alternative to the dmt - mm method for linkage - specific modification has recently been published by us using the combination of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (edc) and 1-hydroxybenzotriazole (hobt) as chemicals for derivatization . Performed in ethanol, the reaction yields highly linkage - specific products when incubating at 37 c for 1 h (lactones for 2,3-linked sialic acids and ethyl esters for 2,6-linked sialic acids), with more resistance to side reactivity (such as amidation) than with dmt - mm . As a result, the method could be performed directly on impure glycan - containing mixtures such as a peptide - n - glycosidase f (pngase f)-released plasma n - glycome . The protocol has been validated for htp application in 96-well plate format, and shows consistent results with ultra - performance liquid chromatography (uplc)-based methods of analysis [fig . 1]. An application has recently been published by bondt et al . Who have studied glycosylation changes on igg fragment antigen - binding (fab) and fc, at different time points during and after pregnancy . By analyzing a total of 576 glycan profile spectra, pregnancy - associated changes were revealed in igg fab glycan sialylation, galactosylation, bisection, fucosylation, and high mannose structures.fig . 12-ab - labeled a3 glycan standard from ludger (cab - a3 - 01) was analyzed by uplc and maldi tof ms after ethyl esterification . A overnight incubation at 37 c of ludger 2-ab - labeled a3 standard with buffer (red), (2,3)-sialidase (green), (2,3/6/8)-sialidase (blue), (2,3/6/8)-sialidase + (1,4)-galactosidase (orange). Separation was performed on a waters acquity uplc h - class system using an acquity beh 1.7 m 2.1 150 mm glycan column . C maldi tof ms spectrum of the 2-ab - labeled a3 standard ([m + na]) after 1 h of ethyl esterification with edc and hobt at 37 c and subsequent hilic purification according to reiding et al . . Profiles obtained from both methods are highly comparable and show similar ratios with regard to sialic acid occupancy and linkage . Hilic peak assignments are based on the exoglycosidase digests, as well as use of internal standards . Structural schemes of glycans are depicted following the cfg notation: n - acetylglucosamine (blue square), fucose (red triangle), mannose (green circle), galactose (yellow circle), n - acetylneuraminic acid (purple diamond). Known n - acetylneuraminic acid linkages are indicated by a left angle (2,3) or right angle (2,6), and otherwise unspecified 2-ab - labeled a3 glycan standard from ludger (cab - a3 - 01) was analyzed by uplc and maldi tof ms after ethyl esterification . A overnight incubation at 37 c of ludger 2-ab - labeled a3 standard with buffer (red), (2,3)-sialidase (green), (2,3/6/8)-sialidase (blue), (2,3/6/8)-sialidase + (1,4)-galactosidase (orange). Separation was performed on a waters acquity uplc h - class system using an acquity beh 1.7 m 2.1 150 mm glycan column . C maldi tof ms spectrum of the 2-ab - labeled a3 standard ([m + na]) after 1 h of ethyl esterification with edc and hobt at 37 c and subsequent hilic purification according to reiding et al . . Profiles obtained from both methods are highly comparable and show similar ratios with regard to sialic acid occupancy and linkage . Hilic peak assignments are based on the exoglycosidase digests, as well as use of internal standards . Structural schemes of glycans are depicted following the cfg notation: n - acetylglucosamine (blue square), fucose (red triangle), mannose (green circle), galactose (yellow circle), n - acetylneuraminic acid (purple diamond). Known n - acetylneuraminic acid linkages are indicated by a left angle (2,3) or right angle (2,6), and otherwise unspecified modification and stabilization of glycoconjugates has recently been attempted by nishikaze et al ., showing a pyaop - based methylamidation of all carboxylic acids present on sialylated glycopeptides . Not only does this derivatization protect the sialylated glycan species from breakdown, maldi - quadrupole ion trap - tof ms fragmentation shows increased structural information as a consequence . Nishikaze and coworkers observed predominantly peptide fragmentation in positive mode collision - induced dissociation ms / ms, while negative mode led to more information on glycan structural features . Using a different glycopeptide derivatization method, amano et al . Modified glycopeptides with 1-pyrenyldiazomethane, also specifically targeting carboxylic acids . Whilst this method leads to enhanced ionization for maldi ms analysis, the subsequent exploration of the fragmentation has shown fragment ions reflecting predominantly sialic acid linkage cleavage . For the htp analysis of underivatized fc igg tryptic glycopeptides, selman et al . Have published a method employing maldi - fourier transform ion cyclotron resonance (fticr)-ms . The advantage of this intermediate - pressure instrument is the tendency for labile substituents to remain intact without chemical stabilization, allowing directly for the analysis of sialylated glycopeptide species . Using 96-well sample preparation, selman et al . Showed a highly repeatable analysis of 384 samples in less than 36 h, comprising all of the steps from capturing of the igg to automatic processing of the spectra . Ms method for measuring igg - fc glycopeptides requiring 16 min analysis time per sample, employing a sheath - flow electrospray setup for enhanced sensitivity and robustness . Large - scale applications of this method have been performed by bondt et al ., analyzing over 1,500 samples within 4 weeks with an internal standard coefficient of variation (cv) below 4% . The results indicated an association between igg galactosylation and improvement of rheumatoid arthritis (ra) during pregnancy . Another application was performed by rombouts et al . Who have used the method to show changes in anti - citrullinated protein antibody fc glycosylation even preceding the onset of ra . Both the fticr and hplc methods separate igg glycopeptides on the basis of single amino acid differences (phenylalanine or tyrosine) allowing subclass - specific glycosylation analysis . An overview of various htp methods for igg glycosylation analysis has been published by huffman et al ., comparing uplc with fluorescent detection, maldi tof ms, multiplexed cge - lif and lc esi another highly sensitive technique for analysis of protein glycosylation utilizes multiple reaction monitoring (mrm) which is an lc mrm is known for its ability to quantify low abundant compounds in highly complex mixtures . For example, glycoproteins from biological fluids like serum or plasma are reduced, alkylated and digested with trypsin and analyzed without further fractionation . In mrm glycans or glycopeptides are scanned in the first quadrupole (q1) for user specified precursor ions and these precursors undergo cid in the second quadrupole (q2) and fragments are produced . Finally the fragments of the precursor ions, known as transitions are scanned in the third quadrupole . Used mrm for quantifying glycopeptides from both model glycoproteins and from depleted human blood serum . [53, 55] also reported the use of mrm for absolute quantitation of igg glycopeptides and also published the use of mrm to study site specific glycosylation alterations for identifying glycan biomarkers . The advantages of the technique are the very high sensitivity which together with the high selectivity allows for the direct analysis of the resulting glycopeptides on the qqq without requiring prior protein enrichment or sample clean - up . A couple of drawbacks to this technique are that (1) mrm analyses are targeted, and analytes / glycopeptide species which are not included in the method set - up cannot be detected by data mining in hindsight . (2) a considerable amount of time and effort need to be invested for assay set - up and for establishing transitions for different analytes . These above mentioned mrm protocols were not adapted to htp sample preparation, but can certainly be implemented for large sample numbers and mrm is envisaged to facilitate glycan biomarker discovery and protein characterization studies in the near future . The analysis of glycosylation is made difficult by technical variation with respect to separation (retention time, m / z values) and intensity (fluorescent signal, ion detection). In particular, the analysis of complex samples is compromised by the presence of the biological matrix that can influence labeling as well as ionization efficacy and migration positions [56, 57]. The use of standards has been found to assist tremendously in identification and quantification of glycans, and many interesting approaches have been shown in the literature to internalize standardization within htp sample analysis . When using a dna analyzer for cge - lif analysis of labeled glycans, the fact can be exploited that the system can analyze fluorescence on different wavelength channels . Using multiplexed cge - lif, co - injection of fluorescently labeled glycans and a base - pair nucleotide ladder fluorescing at a different wavelength was performed allowing for the correction of glycan migration times on the basis of the nucleotide ladder . Glycan standards labeled with heavy isotopes facilitate quantitation during mass spectrometric analysis of derivatized glycans . Tof- and nanospray multistage - ms after labeling with normal 2-aa and a heavy c version of 2-aa . The molecular weight difference between the two labels gives an offset of + 6 da . Mixing various ratios of the differently labeled samples revealed consistent quantitation by mass spectrometry . Using a different labeling chemistry, walker et al . Ms(/ms) analysis and showed quantitative detection after a sample preparation procedure that takes approximately 4 h. as another example, zhang et al . Have presented stable isotopically labeled phenyl-3-methyl-5-pyrazolone at the n - glycan reducing end with quantitation achievable within a 10-fold dynamic range . Variants of these labels have the same mass, but differ in the distribution of heavy isotopes on opposite sides of a bond that readily undergoes fragmentation by cid . Reporter ions in ms experiments can be used for relative quantification, making quantitation of highly complex samples intrinsically more robust than in ms mode quantitation, as the latter method can easily suffer from interfering, overlapping signals . An example of the use of isobaric tags in glycosylation analysis is provided by ahn et al . Who show lc ms / ms data on tryptic glycopeptides derivatized with isobaric tags for relative and absolute quantitation (itraq) at the peptide primary amines . Another version of isobaric tags are tandem mass tags (tmt), which have been used by gong et al . Glycosylamines are generated when pngase f release is performed, but usually hydrolyze to the reducing sugar under most conditions . However, keeping the ph slightly alkaline preserved the glycosylamines and allowed for tmt labeling by n - hydroxysuccinimide ester chemistry . Both itraq and tmt not only facilitate internal standardization, but also allow the discrimination of multiple different samples during a single analysis, speeding up analysis time as a consequence . For example, when a c permethylated glycan standard of known concentration is mixed with a c permethylated glycan sample and analyzed by ms, relative glycan quantitation data can be obtained . Have published the use of c methyl iodide for permethylation, showing expected ratios at various concentrations when comparing to c permethylated human milk oligosaccharides using maldi tof c and c permethylated human igg n - glycan standards developed at ludger are an example of system suitability standards used for relative quantitation [fig when in possession of mass spectrometric instruments with high resolving power, atwood et al . Have shown isobaric variants can also be used for permethylation . Using ch3i and ch2di as methyl donors, a minimal mass difference (0.0029 da) is imparted upon the glycan permethylation site . When analyzed with high resolution instruments such as fticr - ms the variants can be separated, achieving relative quantitation over two orders of magnitude, while a single peak can be generated for the two masses at lower resolution.fig . 2maldi tof ms spectrum of the c and c permethylated human - igg n - glycan standards from ludger (cat #cpm13c - igg-01 and cpmc - igg-01). C was spiked with c on the same sample spot to showcase the comparison of relative quantities of the major igg n - glycans . The mass values shown in the spectra are [m + na] of permethylated glycans, with c permethylated masses in parentheses maldi tof ms spectrum of the c and c permethylated human - igg n - glycan standards from ludger (cat #cpm13c - igg-01 and cpmc - igg-01). C was spiked with c on the same sample spot to showcase the comparison of relative quantities of the major igg n - glycans . The mass values shown in the spectra are [m + na] of permethylated glycans, with c permethylated masses in parentheses various groups have attempted to integrate different parts of the work - up procedure and simplified sample preparation steps . The integration of workflows has the potential to expedite analyses of a wide range of glycans, and the approaches can provide significant advantages in productivity, sensitivity, speed, and efficiency . Blotglyco kits produced by sumitomo bakelite in japan integrate and support an all - in - one solution for automated and htp analysis . Glycoproteins are cleaved with trypsin, glycans are released by pngase f, captured chemoselectively on hydrazide - functionalized beads coupled to 2-ab, and modified at the sialic acids by mtt as described previously . Subsequently, the n - glycans are released from the beads by reduction of sulfide bonds, but by that process retain 2-ab as a label . Using this integration method up to 96 samples can be purified and labeled in about 8 h. a newer method of sumitomo bakelite uses hydrazide beads to capture glycans in a process called glycoblotting, the captured glycans are purified, released from the beads and then labelled with 2ab on a 96 well filter plate . Another integrated system is the agilent mab - glyco chip kit which has been designed for automated characterization of n - glycans from monoclonal antibodies (mab). The system integrates on - chip deglycosylation of the mab, hplc chromatographic separation, quadrupole time - of - flight (qtof)-ms analysis of the released glycans, as well as data analysis by molecular feature extractor which is a part of agilent s data analysis software masshunter . A study comparing on - chip and in - solution deglycosylation methods showed that the analysis time could be reduced from 5 to 6 h to 12 min . Another microchip - based integrated platform is the caliper labchip gxii microchip - ce platform, which uses the caliper profilerpro glycan profiling kit to provide a htp method for analyzing n - glycosylation patterns on mabs and other glycosylated proteins . The analysis is performed on a labchip gxii ce instrument together with an internal standard . By this method, the relative abundance of the major n - glycans found on mabs can be determined in a short span of time, requiring only 90 min for the preparation of 96 samples, although the glycan peak separation is compromised . While the development of htp methods for analysis of glycosylation facilitates the glycomic study of large clinical cohorts, performing manual sample preparation is still a time - consuming process . Additionally, while performing manual work - up is suitable for small sample sets, scaling up to larger sizes may lead to errors and inconsistency, resulting in poor repeatability . This set of circumstances has led to an increasing demand for automation of workflows, with laboratories requiring a system that is simple to operate, has scalable sample preparation and a process that is both repeatable and reliable . Currently, high quality glycomics data can be obtained using liquid handling robots for sample processing, clean - up and sample preparation . Published an automated workflow for igg n - glycan release, hydrazide bead capture of glycans through glycoblotting adapted from the protocol of furukawa et al . Mentioned above, 2-ab labeling, solid phase extraction (spe), lc separation, and quantification . This workflow was developed for the efficient glycoprofiling of bio - therapeutics and clinical samples on the hamilton star liquid handling robot . This approach was tested for the analysis of protein a captured igg derived from cho cells, as well as for the analysis of 100 samples of protein g - captured human igg . The automated glycan preparation was verified with conventional and manual in - gel block release for the human serum igg n - glycans, with cvs below 10% for all major glycan peaks, and with relative ratios in agreement with expectation . A second integrated and automated technology developed specifically for biomolecule sample preparation is the agilent assaymap platform, which is supported by the bravo automated liquid - handling robot . This workflow utilizes prozyme glykoprep - plus chemistry kits, with an optional affinity purification step to capture glycoproteins from cell lysates, cell culture supernatants, or serum . Recoveries in glycoprotein purification approach 100% for most applications, with cvs of 5% . Next, the immobilized glycoprotein samples are treated with pngase f, the glycans fluorescently labeled, and cleaned on hilic cartridges prior to analysis by hplc, ce, or lc ms . The automation platform enables processing of complex samples, increases throughput, decreases variability and decreases assay time from a couple of days to 35 h. however, the cost of the kit could be a limiting factor when analyzing a large number of samples . Another automated analytical workflow supported by the hamilton starlet liquid handling robot has been developed at ludger for htp glycomics for qbd studies . The automated workflow combines glycan release, 2-ab labeling, clean - up, and sample preparation for uplc analysis . A validation study was performed, starting with 16 human igg samples that were released by using pngase f. each sample was divided into four and the four subsets of samples were further divided equally into two 96 well pcr plates . Two operators used the liquid handling robot to label one plate each with 2-ab and cleaned - up using the liquid handling robot . The cvs for the major human igg n - glycan peaks between the different operators were below 5% . In addition, a fully robotized analysis for 48 samples of human igg was performed, starting from the n - glycan release up to uhplc sample preparation, showed cvs less than 5% for all peaks with average areas above 1% [fig . 3].fig . 3 a workflow depicting validation study of semi - automated sample preparation of 48 replicates of human igg samples, comprising digestion by pngase f, 2-ab - labeling, hilic spe enrichment and preparation of samples for injection onto uhplc performed with hamilton starlet liquid handling robot [73, 101, 102]. B typical uhplc chromatogram of 2-ab - labeled human igg glycans prepared using the robot . Analysis of the data from these 48 samples showed cvs less than 5% for all major peaks . Glycan peaks in the chromatogram were assigned by exoglycosidase digestion, standard inclusion, and literature a workflow depicting validation study of semi - automated sample preparation of 48 replicates of human igg samples, comprising digestion by pngase f, 2-ab - labeling, hilic spe enrichment and preparation of samples for injection onto uhplc performed with hamilton starlet liquid handling robot [73, 101, 102]. B typical uhplc chromatogram of 2-ab - labeled human igg glycans prepared using the robot . Analysis of the data from these 48 samples showed cvs less than 5% for all major peaks . Glycan peaks in the chromatogram were assigned by exoglycosidase digestion, standard inclusion, and literature for mass spectrometric analysis of glycopeptides, reusch et al . Have developed a fully automated htp sample preparation method employing the hamilton microlab star to monitor mab igg fc glycosylation by orbitrap esi they studied igg from fermentation supernatant, automating the protein a capturing, tryptic digest, and hilic - spe, showing that the automation produces highly similar results when compared to hilic - hplc analysis of 2-ab labeled glycans, maldi tof ms and high - ph anion - exchange chromatography - pulsed amperometric detection of released glycans, as well as esi - qtof ms of reduced mab fc . The glycopeptide sample preparation performed in this protocol facilitates discrimination between fc and fab portions, allows subclass - specific analysis, and is usable for a wide range of readout methods . Innovations in sample preparation have increased the scope of glycomics considerably, allowing recently, for example, for large - scale gwas [19, 75]. However, increased data production leads to an accompanying need for robust and htp data analysis procedures . The techniques focused on in this review allow the detection of large numbers of samples either by being htp, or by featuring automation leading to decreased hands - on time . Such methods can be used for the discovery of analytes (glycans), but are most helpful for the repeated detection of similar glycosylation features across large cohorts . As a consequence, there is need for software capable of repeated and robust htp feature extraction and quality control . Most analysis platforms and integrated methods have their software solutions, several having been mentioned above, but usually these are closed source, require licensing and may not cater to the functionality required by a user . We recently reported an automated data extraction method for maldi tof ms spectra of n - glycans after sialic acid ethyl esterification . This script requires python, a programming language to be installed (freely available from www.python.org), which is capable of repeatedly integrating a list of glycan compositions from any number of mass spectra . The script calculates the expected glycan masses, allows a variety of modifications, takes the resulting isotopic distributions into account, and performs local background subtraction . As a downside, the program needs already calibrated data, and requires the user to still manually perform quality control . Next to software for repeated analysis of data, several groups have published tools for the interpretation and assignment of glycomic and glycoproteomic data . Notable and freely available examples of de novo interpretation of mass spectrometric data include glyco - peakfinder, glycospectrumscan, glycopep grader, sysbioware, glycominer, and glypid . Interpretation by matching recorded data to a previously annotated database can be performed for mass spectrometric data by glycopeptide search, glycosearchms, and glycopep db, while hplc derived glycan traits can be interpreted by glycoextractor and autogu . Glycomic and glycoproteomic databases have been reviewed by hizal et al . And campbell et al ., of note being the metadatabases glycomedb (containing entries from cfg, kegg, glycosciences.de, bcsdb and carbbank), and unicarbkb (containing entries from glycosuitedb, eurocarbdb, unicarb - db and glycobase). Glycomedb and unicarbkb contain a wealth of theoretical and experimentally observed glycan structures, site - specific information on protein glycosylation, alongside various tools for retrieving and displaying the information . Helpful with the graphical annotation of spectra are the web - based glycanbuilder and drawrings, allowing the facile construction of cfg or oxford type glycan cartoons [93, 94]. Particularly useful for glycan analysis is the desktop application glycoworkbench . The program combines many aspects of glycan annotation and glycan spectral interpretation into a user - friendly package, incorporating glycanbuilder for cartoon annotation, glycopeakfinder for theoretical structure prediction and interpretation, as well as several others for the automatic assignment of spectra, database searching, and many other functions . All tools mentioned here are freely accessible web - based applets, or can be obtained without requiring payment . Analysis of glycosylation is often a major challenge due to the vast macro - heterogeneity of glycoproteins, i.e., a single protein can have a varying number of occupied n- and o - glycosylation sites . Additionally, a single site can be occupied by a variety of glycans, which is known as micro - heterogeneity . Furthermore, the analysis of glycans is complicated by variations in linkage and branching patterns which may lead to multiple isomers . Lc has long been the gold standard for analysis of fluorescently labeled glycans, providing good sensitivity, isomer separation, accurate glycan quantitation, repeatability, and in - depth characterization when supported by exoglycosidase sequencing . Downsides, however, are low sample throughput and relatively high cost per sample when comparing to, for example, ms - based methods of analysis . Ms on the other hand, provides good sensitivity, structural elucidation options via ms experiments, good sensitivity, as well as very high throughput in case of maldi ionization . Downsides here are the limitations in isomer separation and quantitation, and instability of labile groups when no derivatization is performed . Cge - lif supports high sensitivity, high throughput when multiplexed, and low running costs especially when comparing to lc . Unfortunately, databases for cge - lif annotation are small and could be a limiting factor . Hyphenation of glycomic lc and ce methods with online ms detection, optionally in combination with fluorescence detection of labeled glycans, may add confidence in glycan structural assignment as both migration position and (tandem) mass spectrometric information can be combined [97, 98]. However, it should be noted that certainly ce separation of glycans is often compromised by the restrictions faced with ms coupling . Further limitations in lc and ce hyphenation for glycomics are the relatively high costs due to medium sample throughput . As demonstrated in this review, many of the analysis methods are moving towards increasingly htp sample preparation, making use of multiwell formats, employing derivatizations with brief and manageable reaction times, integration of time - consuming steps and providing solutions for rapid analysis of the acquired data . Additionally, this progress towards simpler protocols provides the opportunity for liquid handling robots like the hamilton and bravo types to take over many of the time - consuming and labor- as well as cost - intensive steps . These automated platforms often show similar performance to expert manual labor, while even outperforming manual preparation when large cohorts are measured . A common motif for htp methodology is the inclusion of a solid support glycan binding or adsorption step . When using chemical methods for capturing such as beads containing reactive hydrazide groups, this provides the opportunity for efficient labeling . This kind of integration is an excellent example of development towards shorter and easier protocols . Labeling itself is moving towards the use of heavy isotopes, to allow for reliable quantitation with mass spectrometry . The derivatization steps required for proper analysis of sialylated glycan species, notably permethylation and sialic acid - specific strategies, and several methods have been successful to date . While mass spectrometry is one of the most htp techniques possible for the analysis of glycans and glycoconjugates, it is hampered by its inability to provide quantitative data . This makes the various efforts undertaken for internal standardization, and thereby allowing quantitation, an exciting field to watch . The two main strategies for internal standardization are the use of heavy isotopic variants of glycans as well as the use of isobaric tags with unequal mass distribution . In both cases, the most convenient way to introduce mass differences is by employing derivatization steps already part of an analysis protocol, such as labeling or stabilization . A second strategy would be the spiking of a sample with a known concentration of a standard modified with a heavy isotope, which would also allow for the use of heavy isotopic variants of underivatized glycans . Convenient mass differences for internal standards are a few millidalton showing parallel isotopic distributions on high resolution instruments, and onward from a few dalton to prevent the overlapping of the natural istotopic distribution of glycans and glycopeptides . With increasingly large studies current software for automation of analysis mainly focuses on interpretation rather than repeatable analysis of already interpreted and annotated spectra . There is a clear need for software allowing proper feature extraction, denoising, background subtraction, and most importantly of all, quality control both on individual glycans and complete spectra . Automated interpretation is mostly performed assisted by a predefined set of possibilities, or by matching experimental data to a database . While several programs exist to perform true unassisted de novo annotation, this approach has only shown successful on data that is of high quality . Particularly for hyphenated analysis methods such as lc ms, manual annotation is still the way to go, and is a highly time consuming process . Altogether, the methodological improvements discussed in this article show a clear trend towards more sensitive approaches, with faster analysis, lower costs and less hands - on time . These improvements allow for the increasing translation of glycomic approaches to clinical research, with envisioned applications in patient stratification and personalized medicine.
A 62-year - old man (height, 158 cm; weight, 63 kg; blood type, a) with a history of smoking (35 pack - years) was registered for lung transplantation with a diagnosis of idiopathic pulmonary fibrosis . The patient s status was new york heart association functional class iii, and the patient had lost 5 kg over a 6-month period . Room air arterial blood gas analysis showed an oxygen partial pressure of 59.1 mm hg, partial pressure of co2 was 39.2 mm hg, and oxygen saturation was 89.4% . A pulmonary function test revealed a forced vital capacity of 1.60 l (47% of predicted) and a forced expiratory volume in 1 second of 1.52 l (61% of predicted). Computed tomography revealed subpleural and peribronchovascular reticulation with macrocystic honeycombing in both lungs (fig . 1). Coronary angiography revealed total occlusion at the mid - right coronary artery, and left ventricular ejection fraction of 61% (fig . 2). Therefore, we concluded that the patient needed bilateral lung transplantation and also required coronary artery bypass surgery . Two months after being listed for a lung transplant, a suitable donor lung was matched . The donor was a 49-year - old man (height, 170 cm; weight, 52.1 kg; blood type, a) who had an intracranial hemorrhage . The total lung capacity of the recipient was 4.755 l, while that of the donor was 6.078 l, which was a donor - to - recipient size match of 127.8% . Prior to the lung transplant, the patient s vital signs were stable (heart rate of 98 beats per minute and arterial blood pressure of 115/75 mm hg). After the induction of general anesthesia, a pulmonary arterial catheter was placed via the left internal jugular vein, and the pulmonary arterial pressures were initially 4050/2030 mm hg . The operation was performed as follows: under general anesthesia, a clamshell incision was made and the hila were prepared for lung transplantation . At the same time, the left greater saphenous vein was harvested for coronary artery bypass . Following a minimal dissection in both hila, the saphenous vein was anastomosed to the ascending aorta using a heartstring device (heartstring proximal seal system, guidant, in, usa), and the right coronary artery was bypassed to the right posterior descending artery via the off - pump technique using a tissue stabilizer . A venous cannula was placed in the right femoral vein and an arterial cannula was placed into the right femoral artery . Each side started with bronchial anastomosis, followed by pulmonary venous anastomosis with a side - biting clamp on the pulmonary veins, and ended with pulmonary artery anastomosis . Ecmo was discontinued and decannulated before the patient was transferred from the operating room to the intensive care unit . The total ecmo time was 320 minutes, total operation time was 461 minutes, and total anesthesia time was 565 minutes . On postoperative day (pod) 3, heart rate ranged from 85 to 90 beats per minute, arterial blood pressure ranged from 110 to 120 mm hg/70 to 80 mm hg, and pulmonary arterial pressure ranged from 35 to 37 mm hg/15 to 17 mm hg . The patient required a tracheostomy for recovery due to respiratory failure after tracheal extubation on pod 8 . The patient was transferred to the general ward on pod 16, the tracheostomy tube was removed on pod 30, and discharged on pod 37 in good condition . The presence of comorbidities in patients has previously been considered to be a relative contraindication for lung transplantation . In particular, the first lung transplantation was performed at severance hospital in july 1996, and since then, the number of lung transplantations has increased . At the same time, the inclusion criteria for lung transplantation have been expanded to include patients with higher surgical risk . Whether patients with significant coronary artery disease are suitable candidates for lung transplantation is unclear . Some reports have described the long - term survival outcomes following lung transplantation in cases with revascularized coronary arteries . One report demonstrated that the long - term survival following lung transplantation in patients with significant coronary artery disease was influenced by whether they received coronary revascularization prior to lung transplantation . However, some studies have reported successful results in the management of patients who simultaneously received coronary artery revascularization and lung transplantation . Patel et al . Reported successful simultaneous coronary artery bypass and lung transplantation in a small number of carefully selected patients . However, patients with multivessel disease or left ventricular dysfunction were not considered suitable candidates for lung transplantation . In this case, combined bilateral lung transplantation and coronary artery bypass grafting was performed in a patient who had a single vessel disease with normal left ventricular function . In terms of surgical technique and devices, the off - pump technique was possible for the coronary bypass grafting through a clamshell incision . Using a saphenous vein graft might have enabled coronary artery grafting to be performed without much difficulty through the usual clamshell incision . In korea at present, almost 50% of coronary graft bypass surgeries are performed via off - pump technique . Our previous study indicated that off - pump coronary artery bypass surgeries improved myocardial functioning and favored early and mid - term outcomes in the high - risk group . Both single and bilateral lung transplantation procedure selection should be carefully considering the following factors: recipient, donor, and transplant center . Although single lung transplantation reduced waitlist time and mortality, the long - term outcomes of bilateral lung transplantation were slightly superior . The benefits of bilateral lung transplantation include better lung compliance, imp roved lung volume, and a voidance of native lung disorders . For these reasons, we performed a bilateral lung transplantation . Our center has used both ecmo and conventional cardiopulmonary bypass in the lung transplantation procedure . However, ecmo has been considered the method of choice since 2013 because of its advantages, such as less coagulopathy, less systemic inflammatory response, short postoperative recovery time, and less pulmonary and renal complications . With off - pump coronary artery bypass graft surgery, we could use ecmo for cardiopulmonary support instead of conventional cardiopulmonary bypass . To the best of our knowledge, this case was the first successful bilateral lung transplantation combined with off - pump coronary artery bypass graft surgery in korea, and we reported a satisfactory outcome following surgery.
X - linked dominant chondrodysplasia punctata (cdpx2, omim 302960) is an inheritable systemic disease and is characterized by skeletal, ophthalmologic, and cutaneous manifestations . In the late seventies, rudolph happle reported a female and brilliantly recognized a group of published patients with chondrodysplasia punctata whose mosaic phenotype and sex rate corresponded with an x - linked dominant pattern of inheritance . In 1999, the gene emopamil binding protein (ebp) was identified and associated with cdpx2, so called conradi - hnermann - happle syndrome . The ebp gene is located on the short arm of the x chromosome (xp11.22-p11.23) and spans 7 kb of genomic dna, containing five exons coding a mature transcript of 1 kb which is ubiquitously expressed . This protein consists of 230 amino acids and four transmembrane domains, it has a molecular weight of 27 kda and it is implicated in cholesterol biosynthesis . Cutaneous lesions in cdpx2 usually start as erythematous scaly plaques following the lines of blaschko that fade over time, and some patients may be born with congenital erythroderma . While cdpx2 may exhibit variable phenotypes, psoriasiform skin lesions are exceptional and histopathologic investigations of psoriasiform skin lesions show epidermal hyperproliferation in addition to ichthyotic retention hyperkeratosis . In this report, a case of a 12-year - old girl diagnosed with x - linked dominant chondrodysplasia punctata with a psoriasiform phenotype is reported with genetical confirmation . We discuss here a rare clinical presentation which helps in better depict this rare genodermatosis and on the lack of correlation between phenotype and genotype in this disease . A 12-year - old girl, who had been born with congenital erythroderma, had a 4-month history of linear and whorled inflammatory cutaneous lesions on her trunk, leg, and arms . Extensively distributed thickly adherent scaling had been developed at the age of one, and continued since then . The patient had been born at term following a normal pregnancy and delivery and family history was unremarkable . She displayed growth retardation, bilateral conductive hearing, and unilateral congenital cataract in her right eye . She exhibited short stature with asymmetric shortening of her limbs, being her right arm shorter than her left . Dysmorphic facial features with frontal bossing, flat nasal bridge, and a depressed tip of the nose were observed . Dermatological examination revealed brown streaky hyperkeratotic plaques with thickly adherent scaling located along blaschko's lines . Also, erythematous scaling and guttate lesions were observed on her trunk, arms, and legs . There were patchy areas of scarring alopecia on frontal and temporal regions of the scalp and lateral regions of eyebrows . Dorsal and lumbosacral magnetic resonance imaging showed scoliosis deformity, height losses of the t8 vertebrae left half and l3, l4 vertebral the right halves (tethered cord). Clinical features of the patient with conradi hnermann happle syndrome (x - linked dominant chondrodysplasia punctata) genomic dna of the patient was extracted from peripheral blood leukocytes via a conventional phenol - chloroform method . Exons 1 - 5 and the intronic splicing regions from the ebp gene were amplified by polymerase chain reaction (pcr) as previously described . The reaction was conducted in a 2720 thermal cycler (applied biosystems, foster city, ca, u.s.a . ). Automatic sequencing was performed with the abi prism 310 genetic analyzer (applied biosystems). We identified a previously reported ebp mutation, c. 440 g> a (p.r147h) in the patient, but not in her parents [figure 2]. According to the clinical and laboratory findings, the diagnosis of conradi - hnermann - happle syndrome was established . A skin biopsy of one of the guttate, erythematous, and scaling plaques revealed retention hyperkeratosis, parakeratosis, acanthosis with regular elongation of rete ridges, capillary proliferation, and mononuclear cell infiltration in papillary dermis, being this consistent with a psoriasiform pattern . The pedigree and dna sequencing showed a heterozygous mutation c. 440 g> a resulting in p.r147h in ebp gene in conradi - hnermann - happle syndrome, cutaneous findings are remarkable and characterized by hyperkeratotic lesions on an erythematous background following the blaschko's lines . Bruch et al . Described a unique adult patient presenting both with ichthyotic and pustular psoriasiform lesions together . Later, in a large spanish series, a patient with ichthyosis in a psoriasiform pattern was reported with the genetic analysis showed c. 187c> t (p.arg63x) de novo mutation . We here present an exceptional case of cdpx2 with psoriasiform and ichthyotic skin lesions together in an adolescent, the first case from tunisia . Histopathological features of guttate, erythematous, and scaling plaques followed a psoriasiform pattern . In 1999, the gene ebp was identified and associated with the conradi - hnermann - happle syndrome (xp11.22-p11.23). To date, about 70 different mutations in the ebp gene have been described related with this disease . We found a c.(440 g> a) missense de novo mutation in the ebp gene (p.arg147his), placed in the second endoplasmic reticulum domain (er2) of the protein . The unique case of cdpx2 with a similar phenotype to ours displayed a c.(187c> t), p.arg63x nonsense mutation, which had also been described in two patients with typical skin phenotype of cdpx2 . The c. 440 g seems to be a nucleotide with particularly high mutational susceptibility within the ebp gene, a hot spot . On the other hand, the clinical phenotype associated with this mutation has been variable, which subscribes the lack of correlation between genotype and phenotype in this disease . Although the relationship between the various genotypes and phenotypes in conradi - hnermann - happle syndrome has not been fully elucidated, detailed clinical and molecular analyses are helpful for providing data to be used in genetic counseling . Our case expands the clinical phenotypic variation in cdpx2 and further demonstrates the lack of correlation between genotype and phenotype in this disease . Unique additional clinical manifestations like plaque - type psoriasis may present with different mutations . Conradi - hunermann - happle may represent extensive plaque - type psoriasis in adolescent patients.
Although in most cases (8090%) the ingested foreign body will pass uneventfully through the gastrointestinal tract, sharp foreign bodies such as toothpicks are associated with an increased risk of gastrointestinal perforation and bleeding . The most common causes of the accidental ingestion of toothpicks include alcoholism, rapid food intake and decreased palatal sensitivity . Although the complications caused by swallowed toothpicks are more likely to be detected and resolved by surgical procedure in selected cases when it is possible to locate the position of the toothpick, endoscopic removal can result in the rapid relief of symptoms [3, 4]. A 57-year - old caucasian woman with no previous medical history was admitted to the department of internal medicine, division of gastroenterology, clinical hospital split . She complained of fever and abdominal pain located in right upper quadrant for one week . Vital signs were as follows: blood pressure 120/80 mm hg, pulse rate 100 bpm and core body temperature 39.0c . Laboratory evaluation revealed a wbc count of 12.5 10/l with a left shift and c - reactive protein concentration of 123.6 mg / l while urinalysis as well as other relevant laboratory data were within reference values . Abdominal ultrasound examination was performed because of a clinical presentation mimicking acute cholecystitis, but it was within normal ranges . However, contrast - enhanced computed tomography (ct) scan with three - phase protocol showed an unclearly outlined lesion within the liver parenchyma with a longer diameter of 2.1 cm and which could not be clearly separated from eccentric radiopaque thickening of the stomach antrum in an extension of more than 4 cm (fig . 1, fig . This finding indicated gastroscopy, which revealed a sharp wooden foreign body protruding from the antrum mucosa and the whole wooden foreign body (toothpick) was successfully removed by snare extraction without complications . After the extraction of the foreign body the patient remembered that two weeks earlier when consuming lamb she might possibly have swallowed a toothpick . The patient was also treated with a proton pump inhibitor (pantoprasole), the intravenous administration of crystalloids and ceftriaxone for seven days . Impaction, perforation and obstruction most often occur in areas of acute angulation or physiologic narrowing . It is difficult to estimate the incidence of accidental ingestion of toothpicks, mostly because the literature covering this issue is anecdotal . In contrast to this, it is well known that toothpick lesions to the gastrointestinal tract are often associated with significant morbidity and mortality [2, 3, 4]. Clinical presentation of toothpick gastrointestinal injury includes generalized or local peritonitis, intestinal obstruction or gastrointestinal hemorrhage . In some cases toothpicks migrated outside the gastrointestinal tract and were found in the pleura, pericardium, ureter or bladder [2, 5, 6, 7]. Ingested toothpicks in the esophagus and small intestines are presented by dysphagia or intestinal colic while foreign objects remaining in the stomach are often asymptomatic . Early diagnosis and retrieval of the toothpick is critical for reducing morbidity and mortality [3, 8, 9]. Therefore, we think that a patient presenting with gram - positive bacteremia and no sign of free perforation of the gastrointestinal tract is very illustrative because this demonstrates the necessity of thinking of foreign body ingestion as a cause of obscure bacteremias . We would like to emphasize how important it is to make a very careful anamnesis in order to determine swallowed foreign objects, such as a toothpick, as a rare cause of bacteremia . Moreover, there are no relevant physical or laboratory findings characteristic for a swallowed toothpick . Ct images are useful in acquiring missing clinical information such as toothpick location . In cases without free perforation,
The q - switched nd: yag laser may cause photodisruption at the injury site, which occasionally results in a macular hole . Described 5 eyes of 4 patients with macular holes caused by accidental q - switched nd: yag laser injury . In 1 of these eyes, the macular hole resolved spontaneously . Long - term delayed surgery or observation in these cases may result in a poor or limited outcome [1, 7]. Vitrectomy with gas tamponade has been shown to improve the visual acuity in patients with q - switched nd: yag laser - induced macular holes [2, 5, 6]. We report a case of a macular hole caused by accidental exposure to an nd: yag q - switched laser . We used the following parameters: a fixation target consisting of a single cross, 2 in diameter; a white, monochromatic background at 4 asb (apostilb), a stimulus size goldman iii, with projection time of 200 ms; a customized radial grid of 33 stimuli covering 20 (centered onto the fovea). A 4 - 2 - 1 double staircase strategy was used with an automatic eye tracker that compensates for eye movements . The institutional review board approved the retrospective chart review study . A 31-year - old electronics technician who had undergone bilateral laser - assisted in situ keratomileusis surgery 7 years before accidentally sustained a laser injury to his left eye while repairing and aligning a 1,064-nm nd: yag laser . The q - switched laser pulse energy was approximately 10 mj and the pulse duration was 5 - 20 ns . After a single shot of the laser, he immediately noticed a small central blind spot and floaters in his left eye . At the time of examination within 24 h after injury, his best corrected visual acuity (bcva) was 20/20 in the right eye and 20/200 in the left eye . The examination of the left fundus revealed a marked white juxtafoveal burn with a small retinal hemorrhage and vitreous hemorrhage (fig . 1a). The optical coherence tomography (oct) (stratus oct 3; carl zeiss, dublin, calif ., usa) examination revealed increased thickness in the foveal area and acoustic shadows caused by the vitreous hemorrhage (fig . Fundus examination revealed a macular hole with pigment clumping at its base and elevated edges (fig . In addition, the oct demonstrated a full - thickness macular hole of approximately 820 m in diameter with cystoid changes adjacent to the hole (fig . The mp1-microperimeter demonstrated a retinal sensitivity of 0 db over the macular hole and decreased retinal sensitivity in the adjacent area (fig . The fixation was relatively unstable with 51 and 97% of the preferred fixation points located within a 2- and 4-circle area centered in the fovea, respectively . The fixation target of a single cross was positioned at the preferred fixation point as the foveal center because the center of the foveal avascular zone could not be determined due to the large macular hole . The central fixation location was poor and 46% of the preferred fixation points were located within the 2-circle area centered in the fovea . Surgical intervention was advised because of the unlikely possibility of the spontaneous closure of such a large macular hole . We injected 0.1 ml of indocyanine green (icg) at a concentration of 2.5 mg / ml in the temporal macular region without direct staining of the hole . A bent 25-gauge needle was used to create a small temporal incision 2 disc diameters (dd) away from the fovea . The internal limiting membrane (ilm) was then peeled in the area around the macula (2 dd), and the eye was filled with a 16% c3f8 mixture . The oct showed a marked thinning of the foveal depression and high reflectivity in the subfoveal area with an acoustic shadow (fig . His bcva improved to 20/30 in 3 months and to 20/20 in 12 months postoperatively . The mp1-microperimetry 12 months after surgery demonstrated increased retinal sensitivity in the area of the previous macular hole and its adjacent region (fig . The fixation of the preferred fixation points located within a 2- and 4-circle area centered in the fovea was stable (98 and 100%, respectively). The fixation location was predominantly eccentric and 0% of the preferred fixation points were located within the 2-circle area centered in the fovea . The postoperative preferred fixation position was located at the base of the previous macular hole and was different from the preoperative position (fig . Surgical intervention was advised in our case because of the unlikely possibility of spontaneous closure due to the size of the macular hole (approximately 820 m in diameter) (fig . Yet, an anatomic closure of a macular hole as big in size as noted in our case can be achieved after vitrectomy and peeling of the ilm . However, a marked thinning of the foveal depression was noted after surgery (fig . 1f). The location and severity of the damage of the central fovea tissue may limit the potential improvement in the visual outcome after surgery . In our case, the preoperative visual acuity was 20/60, which may explain the good improvement of visual acuity of 20/20 after 24 months of postoperative follow - up . The amsler grid examination has been used to evaluate the extension of central scotoma and metamorphopsia caused by a laser - induced macular hole [8, 9]. The mp1-microperimeter is a fundus perimeter, which allows for a completely automatic determination of retinal sensitivity and fixation and correlates exactly to the characteristics of the fundus . In this study, mp1-microperimeter after 12 months of postoperative follow - up showed that the retinal sensitivity was increased in the region of the previous macular hole and its adjacent area . The fixation location changed from a poor central fixation to a predominantly eccentric fixation, which may partially be due to the poor preoperative determination of the foveal center and the fixation target positioned at the preferred fixation point as the foveal center preoperatively . However, the postoperative preferred fixation position was different from the preoperative position and was located at the base of the previous macular hole . Our results suggest that vitrectomy can improve the visual function when a macular hole is caused by nd: yag laser injury . The improvement in the visual function includes not only visual acuity but also retinal sensitivity and fixation stability that are obtained by using mp1-microperimeter.
Ectopic gastric mucosa and polyps related to brunner's gland hyperplasia are the most common polyps . Serrated polyps are characterized by infolding of the crypt epithelium, resulting in a saw - tooth appearance . A polyp with serrated morphological features has been classified histologically as hyperplastic polyp in the past . Until the late 1990s, colorectal polyps were generally divided into two major subtypes: hyperplastic polyps and adenomatous polyps . In 1990, serrated came from the observation of the saw tooth - shaped infoldings of the surface and crypt epithelium of these polyps that was similar to that of hyperplastic polyps . Serrated polyps are now classified into 3 distinct types by histologic and genetic characteristics: hyperplastic polyp, sessile serrated adenoma, and transitional serrated adenoma (tsa). A serrated adenoma is a precursor lesion for colorectal carcinoma (crc). The serrated neoplasia pathway has been associated with carcinogenesis of serrated adenoma, which is different from the traditional adenoma - carcinoma sequence [5, 6]. Serrated polyps are commonly found in the colorectum but have rarely been described in other parts of the gastrointestinal tract . Because of the rarity of identifying such a lesion in the small bowel, the natural history, prognosis, and appropriate management recommendations are unclear . Serrated adenomas in the small intestine may represent more aggressive lesions with high malignant potential than those in the colon and rectum, according to some reports . We should consider the existence of serrated adenoma of the duodenum and its higher virulence . We report a case of a slow - growing early adenocarcinoma arising from a traditional serrated adenoma of the duodenum, which was diagnosed and treated by an endoscopic mucosal resection . A 66-year - old man with no significant medical history underwent esophagogastroduodenoscopy (egd) for general examination (fig . 1). He had a 1-cm sized, yamada type iv polyp in the second portion of the duodenum . It was an elevated mucosal lesion with focal white patch and was located at the proximal site of the major papilla . The follow - up egd was done after 2 years (fig . 2). We recommended the patient to resect the polyp; however, he preferred regular follow - up examination every year . There was no change in the shape, size, and pathologic finding of the polyp for two consecutive years . He underwent another egd for general medical check - up 3 years later (5 years after the first detection) (fig . The size of the polyp was slightly increased, but the shape of the polyp was not changed . The lesion was raised by means of a submucosal injection of hypertonic saline tinted with indigo carmine and resected by using a snare . The pathologic result revealed a 0.8 0.5-cm sized, well differentiated tubular adenocarcinoma (fig . 5). Carcinomas are multifocally spread on the traditional serrated adenoma, and the proportion of adenocarcinoma component is approximately 50% . Abdominal pelvic computed tomography and positron emission tomography showed no other solid organ involvement or metastasis . He is now in good health and we will perform surveillance follow - up egd after 1 year . Benign serrated polyps are commonly found in the colorectum but have rarely been described in other parts of the gastrointestinal tract . Histology disclosed an adenomatous growth with unlocked saw tooth - like glands with high - grade dysplasia . Tsas were found in the esophagus, the stomach, the duodenum, the pancreas, and the gallbladder . Increased awareness of the existence of serrated neoplasms in the upper digestive tract rubio's review indicated that 53.4% (n = 39) of the 73 tsas of the upper digestive tract showed a simultaneously growing invasive carcinoma . Following that original publication [13, 14, 15], 35 additional cases of tsa of the duodenum appeared in the literature; 28.6% (n = 10) of the 35 cases showed invasive growth . Of 73 cases of tsa of the upper digestive tract reported in the literature so far, 53.4% (n = 39) had invasive carcinoma . Although the causes for this aggressive behavior remains elusive, it would appear that not only the degree of cellular severity, but also the histological configuration (i.e., with unlocked serrations) might have played a particular role in their virulence . In rosty et al's study, high - grade dysplasia was present in six of the serrated adenomas (46%). One case was an adenocarcinoma resembling a serrated adenocarcinoma of the colorectum, with an adjacent serrated adenoma . This high frequency of high - grade dysplasia suggests that these adenomas may represent aggressive lesions with high malignant potential . Serrated adenomas in the small intestine may represent a distinct morphological subtype of adenoma with a biological significance that is different from those in the colon and rectum . In the current case, it is consistent with other reports that serrated adenoma in the small bowel is more virulent than those in the colon . Tsas of the duodenum should be radically excised, either endoscopically or surgically to rule out the possibility of a synchronously growing invasive adenocarcinoma or to prevent cancer progression . In conclusion, this present case distinctively showed a slow growth but had adenocarcinoma arising from serrated adenoma of the duodenum . We should consider the existence of serrated adenoma of the duodenum and excise it radically owing to its high virulence.
A 16-year - old, castrated male, feline immunodeficiency virus (fiv)-positive, domestic shorthair cat developed multiple skin lesions . Most of these were bowenoid carcinoma in situ and contained dna sequences consistent with felis catus papillomavirus type 2 . Two additional lesions that developed in the skin and subcutaneous tissues between the digital and carpal pads on the left forelimb and right hindlimb were shown by cytology, histology and culture to be caused by prototheca wickerhamii . These lesions failed to improve in response to systemic therapy treatment with itraconazole, but excision by sharp en bloc resection with follow - up oral itraconazole therapy proved curative for one lesion, although the other lesion recurred, necessitating a second surgery . This is only the second reported case of feline protothecosis from australia and the first case that has been cultured and identified to the species level . Also of great interest was the presence of multiple papillomavirus - associated neoplastic lesions, which may have afforded a portal of entry for the algal pathogen and the cat s positive fiv status; the latter might have impacted on both viral and algal pathogenesis by effects on immunocompetence . Prototheca species are currently classified as an achlorophyllic algae, possibly related to chlorella species . They are ubiquitous in the environment and have the potential to cause disease in mammalian hosts, including humans . The first recorded prototheca species infection in a human patient was a focal infection in the foot of a rice worker in sierra leone . Most human infections are focal, caused by prototheca wickerhamii, and probably secondary to local traumatic inoculation of propagules into subcutaneous tissues, including the olecranon bursa . Disseminated infections can occur, however, and comprise perhaps 20% of human cases, typically occurring in immunocompromised individuals . Generally, these disseminated infections are secondary to infection with prototheca zopfii genotype 2 . The first recorded case in the veterinary literature was a case of bovine mastitis in 1952, reported by lerche . Subsequent work has provided detailed information about the epidemiology of this important cause of environmentally associated mastitis, often as case clusters, characteristically in certain geographical regions and associated with deficient husbandry practices, as the problem results from ascending infection from the environment via the teat canal . It is most often recorded in dogs as a disseminated infection following primary infection of the colon (often secondary to a predisposing condition such as granulomatous colitis due to adherent invasive escherichia coli in boxer dogs and french bulldogs) and generally carries a poor prognosis, unless infections are detected while the infection is restricted to the colon, prior to haematogenous dissemination . Disease in cats appears to be extremely rare, with only six cases reported to date . Five of these describe a single focal skin lesion (four caused by p wickerhamii and one secondary to p zopfi genotype 2), presumably of similar pathogenesis to those seen in humans where penetrating injury (usually a cat scratch) introduces infectious propagules from some environmental reservoir . In the single case report with multifocal lesions secondary to p wickerhammii, penetrating local injury would also be suspected, as all lesions were peripheral with no evidence of systemic invasion . Here we report a case in which a cat developed protothecal lesions on two separate paws; the lesions failed to respond to monotherapy with itraconazole, so aggressive surgical excision followed by further itraconazole was undertaken (figure 1). As the cat was infected with feline immunodeficiency virus (fiv) and also had multiple papillomavirus - associated skin lesions, a possible defect in the immune system may have been important in allowing the algal infection to develop . Protothecal lesion left fore: progressive increase in size over 5 months, from (a) when first noticed, through to (b) and then (c), the day of surgery . (d) at 3 months after surgery, the main pad remains, and there is a space where the lesion and the two toes were removed in august 2014, a 16-year - old, castrated male, domestic shorthair cat was presented with multiple skin lesions and weight loss . On physical examination, a heart murmur was heard on auscultation, accompanied by moderate weight loss (8.5 to 6.2 kg over 11 months, without dieting). Skin lesions included a slightly raised plaque (1 cm diameter) on the skin of the dorsal midline between the scapulae and similar smaller masses on the forehead, left medial periorbital skin and the cranial edge of the right pinna . Previous haematological and serum and biochemical tests results from a routine veterinary visit 1 month prior showed a very minimal increase in globulins, mild lymphopenia and total thyroxine concentration within the reference interval (ri). No treatment or further investigations were carried out at that time . At re - examination on october 2014, additionally, there was an ulcerated lesion on the metatarsal pad of the left pelvic limb, accompanied by bilateral enlargement of both popliteal lymph nodes . Meloxicam orally and topical local chloramphenicol / corticosteroid (chloroptson; ceva) ointment were prescribed by the primary veterinarian . The cat re - presented 8 months later (june 2015) with continuing weight loss (now 5.8 kg). The cat had developed a new cutaneous mass between the metacarpal pad and digital pads of the left thoracic limb (figure 1a). A punch biopsy (keys skin biopsy device; 6 mm diameter) of the left forelimb lesion and an excisional biopsy of the entire mass in the dorsal cervical region were performed (http://www.gosh.nhs.uk/health-professionals/clinical-guidelines/skin-biopsy-punch-method) after in - house haematology and biochemistry testing were reported as normal . Histological examination of the skin biopsy from the dorsum showed multiple anatomising, densely packed trabeculae of dysplastic basaloid epidermal cells infiltrating from the ulcerated surface into the dermis . Cells contained vesicular nuclei displaying moderate anisokaryosis, prominent nucleoli, with 23 mitoses per high power field . Nuclei, especially in expanded areas in the epithelium of the superficial portion of the follicle, displayed elongated nuclei orientated in a slanted fashion (windrowing). Squamous cell carcinoma was diagnosed but with features suggesting the lesion may have arisen within a bowenoid in situ carcinoma (figure 2). Squamous cell carcinoma that likely arose within a bowenoid in situ carcinoma: multiple densely packed trabecular of dysplastic epidermal / basaloid like cells infiltrate from an ulcerated surface into the dermis microscopic evaluation of the left forelimb biopsy revealed multifocal to coalescing dermal and subcutaneous infiltrates of macrophages (some multi - nucleate) with lesser numbers of neutrophils . Thus, a diagnosis of pyogranulomatous pododermatitis with large numbers of intralesional bodies consistent with prototheca species was made . The size of the spherical bodies and their shape and morphology was most consistent with p wickerhamii . Multiple - to - coalescing infiltrates of macrophages and neutrophils within the dermis high power view of lesion shown in figure 3 . The arrows indicate some of the spherical bodies within macrophages, and the inset shows staining of these bodies with grocott s methenamine silver stain subsequent cytological examination of a fine - needle aspirate (fna) made from the left forelimb mass, undertaken to improve the morphological detail of the organisms, showed large numbers of macrophages (some multi - nucleate) that contained moderate to sometimes large numbers of thinly encapsulated, spherical, granular organisms (310 m diameter), some of which contained smaller internal spherical structures (figure 5a). Subsequently, a further fna was obtained, sprayed into sterile saline and forwarded to the laboratory for culture . Incubation on sheep blood agar at room temperature and at 37 c produced a moderate pure growth of 2 mm diameter white shiny colonies within 3 days . When examined microscopically, the colonies contained large numbers of spherical, thinly encapsulated spore - like structures (27 m diameter) (figure 5b). Some contained a granular internal structure, although most contained 310 smaller daughter cells (13 m in diameter) that were themselves thinly encapsulated (sporangia). Microscopic morphology, cultural characteristics and failure to metabolise trehalose positively identified the organism as p wickerhamii . Identification using maldi - tof mass spectrometry was also attempted, but was only able to identify the organism as prototheca species, although this technique has been useful for species - level identification . However, dna sequence analysis of the d1/d2 region of the 28s (large subunit) ribosomal dna gene, using published primers and standard sequencing methodology, confirmed the morphological and biochemical identification of the organism as p wickerhamii . (a) fine - needle aspirate preparation stained with diff - quik of left fore lesion showing macrophages, some multinucleate (arrow) containing large numbers of spherical algal bodies . Note spherical spores containing variable numbers of sporangia serial haematology and biochemistry testing was suggested to the owner, and two complete blood counts (cbcs) and serum biochemical examinations, 1 month apart, revealed borderline mature neutropenia (neutrophils 2.2 and 2.5 10/l, respectively; ri 2.512.5 10/l), mild - to - moderate lymphopenia (0.5 and 0.7 10/l, respectively; ri 1.57 10/l), accompanied by a mild - to - moderate hyperglobulinemia (58 and 62 total thyroxine concentration of plasma was within the ri on both occasions (19 and 33 nmol / l, respectively; ri the cat was also fiv antibody elisa - positive and feline leukaemia virus antigen elisa - negative . The animal was placed on a course of cefovecin injections, once fortnightly for 12 weeks . One month after biopsy a further lesion developed on the right hindpaw, on the medial aspect of the skin near digit four . Fna biopsy and culture revealed identical findings to that seen in the left thoracic limb, confirming a second separate focus of pyogranulomatous pododermatitis secondary to p wickerhamii infection . Cytology of the previously noted ulcerated left hind metatarsal pad lesion revealed a suppurative pododermatitis with mixed bacteria and no evidence of protothecal infection . Empirical treatment of the left fore and right hind prototheca - positive lesions commenced with oral itraconazole (sporanox; janssen). Initially, 100 mg total dose (one capsule) was administered once daily (with food), which was reduced to every second day after the animal became lethargic . Despite monotherapy with this triazole antifungal, both algal lesions continued to expand, especially the left forelimb lesion (figure 1). A decision was therefore made to try to eliminate the infections by complete surgical excision of all infected tissues (october 2015) using an oncological - like approach using sharp dissection with en bloc resection of infected tissues, performed by a visiting referral surgeon . The multiple other skin lesions noted previously (that had continued to develop) were also excised at the same time . At surgery, an amorphous hairless and expansile mass (1 cm diameter) was present medial to digit four of the right hindpaw . It was excised with margins of 3 mm by amputation of the digit through the centre of the first phalangeal bone (p1). A fusion podioplasty between digits three and five was performed to prevent digit five from deviating laterally . On the left forepaw, an amorphous mass (2.2 cm diameter) was present with a broad base of attachment to the metacarpal pad, expanding cranially between digits three and four (figure 1c). This lesion was removed by excising the cranial 4 mm aspect of the metacarpal pad, as well as digits two and three through the metacarpophalangeal joints . The distal epiphyses of metacarpal bones two and three were removed with rongeurs to allow the skin of the dorsum of the paw to be directly opposed to the remaining pad without undue tension over bony prominences (figure 1d). Histological examination of resected tissues revealed likely complete removal of the protothecal pyogranulomatous mass (ie, clean surgical margins) from the right hind lesion; however, protothecal organisms could be seen within 2 mm of one surgical margin from the left forelimb lesion, making complete excision of this lesion less confident . Multiple papillomatosis lesions were also excised from the skin of the left hindlimb, the right pinna, the right hindpaw, the lower lip and the dorsal skin between the scapulae and examined histologically . Dna was extracted from formalin - fixed paraffin - embedded tissue shavings from each of the five lesions diagnosed as bowenoid carcinoma in situ, as previously described . To detect the presence of felis catus papillomavirus type 2 (fcapv-2) dna within all primers specific for this pv type were used as previously described . A feline squamous cell carcinoma known to contain fcapv-2 dna was used as a positive control while no template dna was added to the negative controls . Fcapv-2 dna sequences were amplified from the positive control sample and all skin lesions from this patient but not from the negative control sample . Unfortunately, despite this, a recurrence of the lesion on the right hindlimb was confirmed cytologically 8 months after the first surgery and the lesion was surgically removed . Two months later the animal developed ascites, shown to be a borderline modified transudate (protein 22 a cbc revealed persistence of the mild neutropenia and lymphopenia noted previously and the development of a mild anaemia (packed cell volume 22 a limited necropsy examination was performed and a number of organs examined histologically (liver, kidney, spleen, lymph node, pancreas, lung and small intestine). Significant findings were confined to the liver, which showed a moderate diffuse periacinar fibroplasia and hepatocyte atrophy with dilated portal lymphatics . Most previous cases of feline protothecosis have been focal pyogranulomatous dermal and/or subcutaneous lesions with large numbers of intralesional algae . None had evidence of widespread dissemination, although one case did have evidence of infection in the regional draining lymph node . The naso - ocular skin of the cat s head has been affected most commonly, with the location of lesions being mist consistent with a primary cat scratch injury, although distal limb and tail involvement have also been documented . Most cats have been male, consistent with the contribution of fighting and cat scratches to the aetiology of many cases, while there is a wide age distribution (range 315 years). There was no overt evidence of immune suppression in any of these previous feline reports, except for a moderate neutropenia in one case . None of the cats were positive for retrovirus infections, although not all were tested . In the case reported here, the organism was identified as p wickerhamii, which was the cause of all previous cases that were cultured except one . This represents the second case in which there were multifocal lesions with accompanying neutropenia, and in our cat the lesions were restricted to the skin and subcutis of the distal limb, an area where friction and maceration are to be expected . Neutrophils represent an important component of the innate response to saprophytic organisms induced into the subcutis, so the neutropenia might have played a role in terms of predisposing to infection . This cat is the first to be fiv coinfected and the first with concurrent multiple papillomavirus - associated skin neoplasms, some of which may have facilitated penetration of the infectious propagules into host tissues . Traumatic introduction of the organism remains the most likely explanation for the aetiopathogenesis of this infection, as in previous feline cases, and is also likely the cause in focal human cases, as well as bovine mastitis cases where the trauma affects pendulous teats combined with poor environmental hygiene . The lesion in this case failed to respond to systemic treatment with a single fungistatic triazole agent (itraconazole), which is not a surprise as this infection is with algae rather than a true fungus . Ergosterol is far less important as a structural component of algal cell walls than fungal cell wall . Indeed, it is unclear whether the use of this agent impacted at all on the progress of the infection . Such a poor response is consistent with similar cases that have been reported in the feline literature . Perhaps co - administration of terbinafine, amphotericin b (systemically or intralesionally) or all three might have represented a more aggressive and effective approach to the medical management of this patient . In human patients, using a combination of itraconazole and amphotericin b is usually more effective, but use of amphotericin b in cats requires twice - weekly subcutaneous infusions, although intralesional therapy is also possible . Indeed, one could make a strong case that early surgical intervention while lesions are small represents the most cost - effective therapy for most feline patients and the cornerstone for therapy with the goal of curative intent, although consolidation therapy itraconazole and/or amphotericin b may still be important to prevent recurrence of infection due to persistence of algal elements in portions of the wound margin . Resolution was achieved for one lesion here with aggressive oncological - like surgery that required removal of adjacent digits to achieve satisfactory gross surgical margins of 4 mm (see figure 1d). This could be difficult in other locations (eg, lesion on the naso - ocular region, nasal planum). This case also demonstrates the usefulness of fna cytology, which can facilitate early identification of the characteristic morphology of the organism and subsequent culture and species identification . The liver changes detected at necropsy examination were responsible for the development of a modified transudate in the abdominal cavity, and the changes within the liver suggest they have developed over a period of weeks rather than months . The actual cause of the hepatopathy is unknown, perhaps in part because a full necropsy was not permitted . Itraconazole can have toxic effects on hepatocytes, although there were never any elevations in alanine transferase activity (a hepatocyte cytosolic - specific enzyme in cats) on repeated sampling of this cat before and during treatment . P wickerhamii is a rare cause of pyogranulomatous dermatitis in felines, but diagnosis is facilitated by cytological examination . Lesions may be treated successfully with full surgical excision, if location and size of lesion allow . This case report also suggests that immune suppression and pre - existing ulcerative lesions may contribute to local infection in some cats.
A 32-year - old healthy caucasian lady presented complaining of recent deterioration of vision in her left eye . At presentation, her best corrected visual acuity (bcva) was 20/20 in her right eye and counting fingers in her left eye (le). Fundus examination and fluorescein angiography revealed findings consistent with arteriovenous communications of the retina or racemose hemangioma, in the posterior pole of the le with the presence of macular ischemia . Complete and systemic examination was unremarkable, excluding the possibility of wyburn - mason syndrome . Eight years after presentation, findings and bcva in the le have remained stable, with no extension of the retinal ischemia or development of neovascularization . Although extensive retinal ischemia has been reported to result in complications such as retinal or iris neovascularization, in our case the macular ischemia has not expanded further over a period of 8 years . However, due to this macular ischemia the patient unfortunately lost her central vision . Racemose hemangioma is rare . The development of extensive retinal ischemia including macular ischemia resulting in rubeosis has been reported . We describe a case of racemose hemangioma which spontaneously developed macular ischemia alone, resulting in poor visual acuity . This finding has remained stable over a follow - up period of 8 years with no further complication . A 32-year - old healthy caucasian lady presented complaining of recent deterioration of vision in her left eye (le). At presentation, her best corrected visual acuity (bcva) was 20/20 in her right eye and counting fingers in her le . The anterior segment and intraocular pressures were normal in both eyes . Fundus examination and fluorescein angiography revealed findings consistent with arteriovenous communications of the retina or racemose hemangioma, in the posterior pole of the le with the presence of macular ischemia (figures 1 and 2). Complete and systemic examination including mri scan was unremarkable, excluding the possibility of wyburn - mason syndrome with arteriovenous malformations of the optic nerve and midbrain . Eight years after initial presentation, findings and bcva in the le have remained stable, with no extension of the retinal ischemia or development of neovascularization . The lesions have been reported either to remain static or regress12 or to enlarge gradually . Vision may be affected directly due to macular involvement or by producing hemorrhage or exudation.3,4 based on archer et al the angioma in our case was grade 3, although there were no systemic findings.5 we present a case of racemose hemangioma which spontaneously developed macular ischemia . Although extensive retinal ischemia has been reported to result in complications such as retinal or iris neovascularization,1 in our case the macular ischemia has not expanded further over a period of 8 years . However, due to this macular ischemia the patient unfortunately lost her central vision . It has been postulated that either an enlarged malformation using part of the blood supply of the retina may cause ischemia, or there was a partial thrombosis which caused circulatory stasis within the lesion.6,7
Receptor tyrosine kinases (rtks) are a class of protein kinases that play a critical role in the development and progression of various types of cancer . Recently, approximately 20 different rtk classes have been identified, with axl belonging to one of these classes . The axl receptor tyrosine kinase was isolated as a transforming gene from primary human myeloid leukemia cells . Axl is implicated in vascular remodeling, regulation of smooth muscle cells, and migration of endothelial cells . In recent years, the significant role of axl in tumor initiation and metastases has been reinforced by the fact that axl is overexpressed in multiple cancer types including prostate, breast, lung, gastric, glioblastoma, and kaposi sarcoma . Several studies also indicate that the expression level of axl is highly correlated with lung tumor progression and invasiveness of breast cancer cells . In addition, axl is identified as a potential therapeutic target for overcoming egfr inhibitor resistance and for lapatinib and trastuzumab resistance in breast cancer cells . In order to hasten the pace of developing anti - axl based cancer therapy for clinical trials, it is desirable to establish effective methods to quantify axl expression in vivo . Molecular imaging techniques have been widely used for diagnosis and therapy management through evaluation of molecular marker and receptor expression in vivo . In particular, positron emission tomography (pet) has gained a remarkable amount of attention due to its quantitative imaging properties and high sensitivity . In fact, there is a considerable amount of interest in development of imaging probes for pet that can serve as companion diagnostics for novel therapeutics . However, to the best of our knowledge, no pet probe targeting axl has been reported in previous literature . Recently, monoclonal antibodies of murine origin that are specific for axl have been developed . One of these antibodies, m173, appears to bind to the first fibronection domain . To enhance clinical application, humanized 173 (h173) because of the strong and specific binding of h173 to axl, we hypothesized that radiolabeled h173 could depict the axl receptor distribution in vivo by pet . The data obtaining by pet imaging could be used to confirm the presence of axl, which would be important clinical information in determining the utility of axl - targeted chemo- and radiotherapy in receptor positive patients . In this study, we radiolabeled h173 with cu to create an antibody based pet probe to noninvasively quantify axl expression in vivo . Human small cell lung cancer a549 cell line and nonsmall cell lung cancer cell line nci - h249, obtained from american type culture collection (manassas, va), were grown in rpmi-1640 supplemented with 10% fetal bovine serum . For facs analysis, aliquots of cells were blocked with 10% normal goat serum, incubated with anti - axl primary antibody h173 (kindly provided by vasgene therapeutics inc ., los angeles, ca) produced using composite human antibody technology and goat antihuman alexa fluor 488 (invitrogen, paisley, scotland) in sequence . Subsequently, cells were measured by flow cytometry (cyan analyzer, beckman coulter). Proteins extracted from a549 and nci - h249 tumor cells were fractionated using 420% sds - page (bio - rad). After protein transfer, polyvinylidene difluoride membrane was blocked with 5% nonfat dry milk, probed with anti - axl primary antibody (cat #8661, cell signaling technology, beverly, ma) and secondary antibody, and developed . H173 was conjugated with dota - nhs synthesized in situ (molar ratio, 1:20) through amino groups to form dota - h173 . Negative control antibody, human normal immunoglobulin g (higg), was purchased from rockland (gilbertsville, pa). Control probe dota - higg was also synthesized using the same procedure . After cu (purchased from washington university, st . Louis) labeling, probes were used for further in vitro and in vivo experiments . Axl binding activity of dota - h173 and dota - higg was performed through a bead - based binding assay with axl - alkaline phosphatase (ap) (kindly provided by vasgene therapeutics inc . Cell uptake of probes in a549 and nci - h249 tumor cells was performed as described previously . 5 10 cells were suspended in 200 l of complete cell culture media, and 37 kbq of cu - dota - h173 and cu - dota - higg was added . After 1.5 h of incubation, unbound probes were removed by washing twice with cold pbs . Finally, cells were sedimented by centrifugation, and the radioactivity in each cell pellet was counted . All animal experiments were performed under a protocol approved by the university of southern california institutional animal care and use committee (iacuc). To establish a lung tumor xenograft model, 2 10 of a549 or nci - h249 cells were subcutaneously injected in the right shoulder of nude mice as previous reported . The tumor - bearing mice were injected with 3.77.4 mbq of cu probes via tail veins . For each probe, 3 randomly selected mice were used . Multiple static scans were obtained at 3, 16, 28, and 45 h postinjection (p.i . ). Pet imaging and analysis were conducted by using a siemens micropet r4 rodent model scanner as described previously . Antibody distribution was localized by using secondary antibody goat antihuman alexa fluor 568 (invitrogen, paisley, scotland). All of the quantitative data are given as means sd of three independent measurements . H173 and higg were conjugated with cu chelator dota through amino groups, which lead to dota - h173 and dota - higg . After cu labeling, the radiochemical yields for cu - dota - h173 and cu - dota - higg were 44.5% and 57.6%, respectively . The specific activity of cu - dota - h173 and cu - dota - higg was estimated to be 1.482.96 gbq / mg antibody . To investigate the influence of dota conjugation on axl binding ability, a binding activity assay was conducted . Axl binding activity was preserved with dota - h173 (98.27% 1.29%). On the contrary, we used a549 and nci - h249 human lung cancer cell lines for this study . A western blot was performed to detect axl expression in these two cell lines . As shown in figure 1a, a549 overexpressed axl, while nci - h249 was negative . Fluorescence - activated cell sorting (facs) data demonstrated that the percentage of axl positive in a549 and nci - h249 was 84.40 1.56% and 2.43 0.27%, respectively (figure 1b). Cell uptake study was also conducted (figure 1c). In a549 cells, the cell uptake of cu - dota - h173 (1.96 0.10%) was significantly higher than cu - dota - higg (0.36 0.04%) (p <0.05). The cell uptake of both cu - dota - h173 (0.32 0.05%) and cu - dota - higg (0.30 0.05%) in nci - h249 cells was low and showed no significant difference between them (p> 0.05). The above in vitro data demonstrated that cu - dota - h173 probe was axl - specific . (a) western blot of axl in a549 and ncl - h249 tumor cells . (b) facs analysis of a549 and ncl - h249 tumor cells using h173 as the primary antibody . (c) cell uptake assay of cu - dota - higg and cu - dota - h173 on a549 and ncl - h249 tumor cells (n = 3, mean sd). The in vivo micropet imaging study was performed on both a549 and nci - h249 tumor xenograft mice after the injection of cu - dota - h173 or cu - dota - higg . Representative decay - corrected coronal images are shown in figure 2 . At an early time point, both cu - dota - h173 and cu - dota - higg demonstrated high heart uptake because of the relative long circulation half - life of antibodies in vivo . Similarly, the liver also had relatively high uptake, which could be attributed to the nonspecific uptake of tracers through the reticular - endothelial system . As for other organs, such as the lung, their uptake was not significantly different from the background such as muscle . The uptake in the tumor or other organs was calculated from the roi analysis and shown in figure 3 . The a549 tumor uptake of cu - dota - h173 was 5.02 1.00, 8.32 0.89, 11.37 1.53, and 13.37 1.53% id / g (percentage of injected dose per gram of tissue) at 3, 16, 28, and 45 h p.i ., respectively . The nci - h249 tumor uptake of cu - dota - h173 was 3.66 0.64, 4.52 1.08, 2.65 0.36, and 2.21 0.30% id / g at 3, 16, 28, and 45 h p.i . Cu - dota - h173 uptake in a549 tumors was significantly higher than in nci - h249 tumors (p <0.05, figure 3d) at 16, 28, and 45 h time points . The a549-tumor to lung ratios were 1.21 0.25, 2.42 0.36, 4.35 1.80, and 6.53 2.10% id / g at 3, 16, 28, and 45 h p.i . Cu - dota - higg was also imaged in a549 tumor models to validate the target specificity of cu - dota - h173 . The a549 tumor uptake of cu - dota - higg at all time points examined was comparatively low (3.26 0.94, 3.59 0.54, 2.09 0.55, and 1.76 0.58% id / g at 3, 16, 28, and 45 h p.i ., respectively), which is similar to the muscle uptake (4.28 1.23, 3.35 0.65, 2.52 0.98, and 1.69 0.66% id / g at 3, 16, 28, and 45 h p.i ., decay - corrected whole - body coronal micropet images from a static scan at 3, 16, 28, and 45 h p.i . Of (a) (b) cu - dota - h173 into ncl - h249 tumor bearing mice, and (c) cu - dota - higg into a549 tumor bearing mice . Major organ radioactivity accumulation quantification from a static scan at 3, 16, 28, and 45 h p.i . Of (a) (b) cu - dota - h173 into ncl - h249 tumor bearing mice, and (c) cu - dota - higg into a549 tumor bearing mice . (d) time course of a549 and ncl - h249 tumor uptake of cu - dota - h173 from 3 to 45 h p.i . Probe distribution analysis in tumor tissues was conducted through immunofluorescence staining . As shown in figure 4, more h173 accumulated in axl - positive a549 tumors than higg control . In axl - negative nci - h249 tumors therefore, both in vitro and in vivo data indicated that cu - dota - h173 could be applied for noninvasive imaging of axl expression . Up until now, 20 distinct subfamilies of rtks have been found and categorized according to their identities in their amino acid sequence and structural similarities in their extracellular regions . One of these, the subfamily tam, is composed of axl, sky, and mer . Axl and its ligand, gas6, have been found to play a pivotal role in the survival and metastasis of multiple cancers and other diseases . In this study, the novel cu - labeled h173 antibody was synthesized and characterized to demonstrate that imaging axl expression with pet is possible . Attachment of cu to a targeting molecule required the use of a bifunctional chelator (bfc). In our experiment, binding activity assay showed that dota - h173 preserved 98.27% 1.29% axl binding activity . Facs (figure 1b) and the cell uptake study (figure 1c) further confirmed that h173 could bind to axl - positive a549 lung cancer cells, but not to axl - negative nci - h249 tumors, indicating that h173 antibody was axl - specific . Western detects both cell surface and cytoplasm axl while flow cytometry detects only cell surface axl . Therefore, the receptor expression difference in flow cytometry was not as drastic as the western data . The in vivo micropet imaging study was performed on both a549 and nci - h249 tumor xenograft mice (figures 2 and 3). Cu - dota - h173 uptake in a549 tumors increased with time, and a better tumor - to - background contrast was displayed at late time points (28 h, 45 h p.i . ). Both active uptake (contributed by h173-axl interaction) and passive uptake (contributed by the enhanced permeability and retention effect) in contrast, tumor accumulation of cu - dota - higg was only caused by passive uptake . The a549 tumor uptake of cu - dota - higg at all time points examined was comparatively low . Thus, the target specificity of cu - dota - h173 was confirmed . On the contrary, cu - dota - h173 uptake in nci - h249 tumors was significantly lower than in a549 tumors (p <0.05, figure 3d). As h173 only recognizes human axl, it does not target murine vasculature, which also expresses axl in tumor xenograft mouse models, according to the published studies . Therefore, the difference in tumor uptake should reflect the axl expression levels of different lung tumor cells . We would like to point out that tumors without axl expression could still show some tracer uptake due to the presence of nonspecific accumulation . In order to accurately reflect axl receptor expression level with immunopet, the contribution from passive targeting needs to be considered during data analysis . However, in humans, use of cu - dota - h173 for axl - targeted imaging might provide higher contrast from surrounding tissues due to the fact that it recognizes both tumor cells and associated vasculature . In addition, highly selective localization of h173 to the tumor bed could lead to applications with therapeutic radionuclides, such as y, lu, or bi for targeted radiation therapy . We have developed a receptor - targeted pet probe for detection of axl - positive tumors . This study shows that cu - dota - h173, based on a humanized axl - specific monoclonal antibody, displays high target specificity both in vitro and in vivo . The positive correlation between axl expression levels and tumor invasiveness in multiple cancer types makes cu - dota - h173 potentially clinically useful for staging axl - positive cancers and managing patients with axl - targeted therapy.
Data was provided by a larger, ongoing, longitudinal study of rape victims who presented at a center for rape victims (crv) at the university hospital, aarhus, denmark . Four hundred and eighty - four participants were administered the asds between two and three weeks post - rape . However, eight cases were missing> 20% and thus were excluded from the analysis, leaving an effective sample size of 476 . Three hundred and twenty - six (68.5%) participants met the dsm - iv criteria for each of the four symptom clusters . Mean scores for symptom clusters were 17.90 (sd=3.2, range: 11 to 25), 12.87 (sd=3.3, range: 6 to 20), 13.29 (sd=3.2, range: 620), and 21.35 (sd=4.7, range: 10 to 30), dissociation, intrusion, avoidance, and arousal respectively . 60) and all but one participant was female . For further details about the study the second sample comprised 152 bank employees exposed to bank robberies committed in denmark between september 2008 and march 2010 . Participants were recruited through a network of crisis intervention specialists contracted with bank organizations in denmark . However, three cases were missing> 20% and thus were excluded from the analysis, leaving an effective sample size of 149 . A total of 25 (16.8%) participants met the dsm - iv criteria for each of the four symptom clusters . Mean scores for symptom clusters were 16.68 (sd=3.6, range: 11 to 23), 13.56 (sd=3.3, range: 9 to 20), 10.32 (sd=2.8, range: 6 to 16), and 21.92 (sd=5.3, range: 10 to 30) for dissociation, intrusion, avoidance, and arousal respectively . Data was provided from a national study of bank robberies committed in denmark from april 2010 to april 2011 . The study was conducted in collaboration between the danish bankers association, all danish banks, and the university southern of denmark . A total of 450 participants were administered the asds of which 53 (11.8%) participants met the dsm - iv criteria for each of the four symptom clusters . Mean scores for symptom clusters were 17.02 (sd=2.8, range: 12 to 24), 12.36 (sd=3.4, range: 7 to 20), 10.11 (sd=3.2, range: 618), and 20.25 (sd=4.4, range: 1030) for dissociation, intrusion, avoidance, and arousal respectively . (in press). Collectively, a total of 404 participants met the dsm - iv criteria for each of the four asd clusters (please refer to the measure section for further detail). The mean scores on the asds dissociative, intrusion, avoidance, and arousal subscales were 17.7 (sd=3.2, range: 11 to 25), 16.3 (sd=3.6, range: 9 to 25), 12.7 (sd=3.4, range: 6 to 20), and 21.2 (sd=4.7, range: 1030), respectively . The asd latent structure was assessed using the danish version of the acute stress disorder scale in all three samples (asds; bryant et al ., 2000).the asds is a 19 item self - report scale with four subscales assessing the four separate symptom clusters; dissociation, re - experiencing, avoidance, and arousal as specified by the dsm - iv . Questions are answered on a five - point likert scale (1 = not at all, 5 = very much). The asd symptom clusters were met if the participants endorsed at least one re - experiencing symptom, one avoidance symptom, and one arousal symptom in addition to at least three dissociative symptoms, all indicated by item scores 3 on the asds . Previous studies have used this procedure and have reported good reliability, with reliability coefficients of .85, .90, .93, and .96 (armour et al ., 2011; elklit & christiansen, 2010; hansen, in press; hansen & elklit, 2011) for the total scale . The reliability coefficient in the current study for the full combined sample was satisfactory (total scale=.76). Nominal amounts of item - level missing data were present (<4 items) thus maximum likelihood (ml) estimation procedures were implemented (graham, 2009) prior to the merging of the three separate samples . All analyses were conducted using mplus 6 software (muthn & muthn, 2010). We specified and estimated four competing models (see table 1 . For asds item distributions). Assumptions of univariate (no skewness / kurtosis values> 1.35) and multivariate (mahalanobis d) were met thus we used maximum likelihood estimation in cfa . When specifying the models, the first item in each latent factor was fixed to 1 . In addition, we allowed all factors to correlate but we did not allow for correlated errors . Thus, we inspected the chi - square (), the comparative fit index (cfi: bentler, 1990), tucker lewis index (tli: tucker & lewis, 1973), the root mean square error of approximation (rmsea: steiger, 1990), and the bayesian information criterion (bic: schwarz, 1978). Adequate model fit is demonstrated by a non - significant, cfi's and tli's between .90 and .94 and rmsea's <.08 . Excellent model fit is indicated by cfi's and tli's>.95, and rmsea's <.06 (hu & bentler, 1999). Raftery (1995) reported that superior model fit is indicated by a 10-point bic difference, which represents a 150:1 likelihood that the model with the lower bic value fits best (p <.05). Additionally, given that fan and sivo (2009) reported that it is inaccurate to compare nested models on the basis of fit indices alone, we compared nested models using chi - squared difference tests by employing the mplus difftest function (muthn & muthn, 2006). As chi - square difference testing cannot be conducted for non - nested models comparisons data was provided by a larger, ongoing, longitudinal study of rape victims who presented at a center for rape victims (crv) at the university hospital, aarhus, denmark . Four hundred and eighty - four participants were administered the asds between two and three weeks post - rape . However, eight cases were missing> 20% and thus were excluded from the analysis, leaving an effective sample size of 476 . Three hundred and twenty - six (68.5%) participants met the dsm - iv criteria for each of the four symptom clusters . Mean scores for symptom clusters were 17.90 (sd=3.2, range: 11 to 25), 12.87 (sd=3.3, range: 6 to 20), 13.29 (sd=3.2, range: 620), and 21.35 (sd=4.7, range: 10 to 30), dissociation, intrusion, avoidance, and arousal respectively . 60) and all but one participant was female . For further details about the study the second sample comprised 152 bank employees exposed to bank robberies committed in denmark between september 2008 and march 2010 . Participants were recruited through a network of crisis intervention specialists contracted with bank organizations in denmark . However, three cases were missing> 20% and thus were excluded from the analysis, leaving an effective sample size of 149 . A total of 25 (16.8%) participants met the dsm - iv criteria for each of the four symptom clusters . Mean scores for symptom clusters were 16.68 (sd=3.6, range: 11 to 23), 13.56 (sd=3.3, range: 9 to 20), 10.32 (sd=2.8, range: 6 to 16), and 21.92 (sd=5.3, range: 10 to 30) for dissociation, intrusion, avoidance, and arousal respectively . Data was provided from a national study of bank robberies committed in denmark from april 2010 to april 2011 . The study was conducted in collaboration between the danish bankers association, all danish banks, and the university southern of denmark . A total of 450 participants were administered the asds of which 53 (11.8%) participants met the dsm - iv criteria for each of the four symptom clusters . Mean scores for symptom clusters were 17.02 (sd=2.8, range: 12 to 24), 12.36 (sd=3.4, range: 7 to 20), 10.11 (sd=3.2, range: 618), and 20.25 (sd=4.4, range: 1030) for dissociation, intrusion, avoidance, and arousal respectively . (in press). Collectively, a total of 404 participants met the dsm - iv criteria for each of the four asd clusters (please refer to the measure section for further detail). The mean scores on the asds dissociative, intrusion, avoidance, and arousal subscales were 17.7 (sd=3.2, range: 11 to 25), 16.3 (sd=3.6, range: 9 to 25), 12.7 (sd=3.4, range: 6 to 20), and 21.2 (sd=4.7, range: 1030), respectively . Data was provided by a larger, ongoing, longitudinal study of rape victims who presented at a center for rape victims (crv) at the university hospital, aarhus, denmark . Four hundred and eighty - four participants were administered the asds between two and three weeks post - rape . However, eight cases were missing> 20% and thus were excluded from the analysis, leaving an effective sample size of 476 . Three hundred and twenty - six (68.5%) participants met the dsm - iv criteria for each of the four symptom clusters . Mean scores for symptom clusters were 17.90 (sd=3.2, range: 11 to 25), 12.87 (sd=3.3, range: 6 to 20), 13.29 (sd=3.2, range: 620), and 21.35 (sd=4.7, range: 10 to 30), dissociation, intrusion, avoidance, and arousal respectively . 60) and all but one participant was female . For further details about the study the second sample comprised 152 bank employees exposed to bank robberies committed in denmark between september 2008 and march 2010 . Participants were recruited through a network of crisis intervention specialists contracted with bank organizations in denmark . However, three cases were missing> 20% and thus were excluded from the analysis, leaving an effective sample size of 149 . A total of 25 (16.8%) participants met the dsm - iv criteria for each of the four symptom clusters . Mean scores for symptom clusters were 16.68 (sd=3.6, range: 11 to 23), 13.56 (sd=3.3, range: 9 to 20), 10.32 (sd=2.8, range: 6 to 16), and 21.92 (sd=5.3, range: 10 to 30) for dissociation, intrusion, avoidance, and arousal respectively . Data was provided from a national study of bank robberies committed in denmark from april 2010 to april 2011 . The study was conducted in collaboration between the danish bankers association, all danish banks, and the university southern of denmark . A total of 450 participants were administered the asds of which 53 (11.8%) participants met the dsm - iv criteria for each of the four symptom clusters . Mean scores for symptom clusters were 17.02 (sd=2.8, range: 12 to 24), 12.36 (sd=3.4, range: 7 to 20), 10.11 (sd=3.2, range: 618), and 20.25 (sd=4.4, range: 1030) for dissociation, intrusion, avoidance, and arousal respectively . Collectively, a total of 404 participants met the dsm - iv criteria for each of the four asd clusters (please refer to the measure section for further detail). The mean scores on the asds dissociative, intrusion, avoidance, and arousal subscales were 17.7 (sd=3.2, range: 11 to 25), 16.3 (sd=3.6, range: 9 to 25), 12.7 (sd=3.4, range: 6 to 20), and 21.2 (sd=4.7, range: 1030), respectively . The asd latent structure was assessed using the danish version of the acute stress disorder scale in all three samples (asds; bryant et al ., 2000).the asds is a 19 item self - report scale with four subscales assessing the four separate symptom clusters; dissociation, re - experiencing, avoidance, and arousal as specified by the dsm - iv . Questions are answered on a five - point likert scale (1 = not at all, 5 = very much). The asd symptom clusters were met if the participants endorsed at least one re - experiencing symptom, one avoidance symptom, and one arousal symptom in addition to at least three dissociative symptoms, all indicated by item scores 3 on the asds . Previous studies have used this procedure and have reported good reliability, with reliability coefficients of .85, .90, .93, and .96 (armour et al ., 2011; elklit & christiansen, 2010; hansen, in press; hansen & elklit, 2011) for the total scale . The reliability coefficient in the current study for the full combined sample was satisfactory (total scale=.76). Nominal amounts of item - level missing data were present (<4 items) thus maximum likelihood (ml) estimation procedures were implemented (graham, 2009) prior to the merging of the three separate samples . All analyses were conducted using mplus 6 software (muthn & muthn, 2010). Assumptions of univariate (no skewness / kurtosis values> 1.35) and multivariate (mahalanobis d) were met thus we used maximum likelihood estimation in cfa . When specifying the models, the first item in each latent factor was fixed to 1 . In addition, we allowed all factors to correlate but we did not allow for correlated errors . Thus, we inspected the chi - square (), the comparative fit index (cfi: bentler, 1990), tucker lewis index (tli: tucker & lewis, 1973), the root mean square error of approximation (rmsea: steiger, 1990), and the bayesian information criterion (bic: schwarz, 1978). Adequate model fit is demonstrated by a non - significant, cfi's and tli's between .90 and .94 and rmsea's <.08 . Excellent model fit is indicated by cfi's and tli's>.95, and rmsea's <.06 (hu & bentler, 1999). Raftery (1995) reported that superior model fit is indicated by a 10-point bic difference, which represents a 150:1 likelihood that the model with the lower bic value fits best (p <.05). Additionally, given that fan and sivo (2009) reported that it is inaccurate to compare nested models on the basis of fit indices alone, we compared nested models using chi - squared difference tests by employing the mplus difftest function (muthn & muthn, 2006). As chi - square difference testing cannot be conducted for non - nested models comparisons were based on differences in bic values . The mean asds total score for the combined sample who met the dsm - iv diagnostic criteria for all four symptom clusters (n=404) was 64.5 (sd=10.1, range: 4090). Notably, the values of all models were statistically significant . However, this should not lead to rejection of models given that chi - square values tend to become non - significant when assessing model fit with samples which have> 200 participants (current n=404) (schumacher & lomax, 1996). None of the models reached the recommended values of the cfi and tli for adequate (between .90 and .95) or excellent (>.95) fit . The rmsea failed to reach recommended values for adequate or excellent fit for model 1; however rmsea values for model 2 and 3 indicated adequate model fit, whereas the rmsea for model 4 indicated excellent model fit . Fit indices for the four alternative asd models, chi - square; rmsea, root mean square error of approximation; ci, confidence interval; cfi, comparative fit index; tli, tucker as stated above we compared nested models using chi - squared difference tests by employing the mplus difftest function (muthn & muthn, 2006). Model 2 did not fit significantly better than model 3, change (3, n=404) = 10.17, p=.0172 . However, model 4 fit significantly better than both model 2, change (4, n=404) = 71.29, p<.000 and model 3, change (7, n=404) = 77.33, p<.000 (cf . Thus, model 4 provided optimal fit to the data compared to alternative models in the current study . Therefore, the corresponding standardized factor loadings for the asd dysphoric arousal replication model can be found in table 3 . Standardized factor loadings (standard errors) for the asd dysphoric arousal model note: all factor loadings were statistically significant (p<.05). Inter - factor correlations for the dysphoric arousal model of asd the correlation which was not significant (p<.001). The present study is the first study to assess a replication of ptsd's newest five - factor dysphoric arousal model using asd data . A competing models approach was implemented in which the asd dysphoric arousal model was compared to three alternative asd models, in a combined heterogeneous clinical trauma sample . Akin to a burgeoning literature base in relation to the latent structure of ptsd (reviewed in pietrzak et al ., 2012) results suggest that the latent structure of asd, measured by the asds, is best described as a five - factor structure separating the arousal factor into two separate factors of dysphoric arousal and anxious arousal . Thus, it appears that watson's (2005) distinction between arousal related to general distress (i.e., dysphoric arousal) and arousal related to fear - based symptoms (i.e., anxious arousal) in relation to ptsd also holds for acute posttraumatic symptoms . Hence, it is possible that arousal symptoms play a similar role in both early and long term posttraumatic responding . The pronounced role of arousal symptoms in posttraumatic symptomatology is highlighted in schell, marshall, and jaycox's (2004) prospective study of the relationship between ptsd symptom clusters in victims of community violence . 2004) found that arousal strongly influenced the other symptom clusters, but at the same time arousal was generally not influenced back by the other symptom clusters . Furthermore, arousal was found to be the best predictor of subsequent ptsd symptom severity compared to alternative ptsd symptom clusters . Indeed, schell et al . (2004) found that those with the most pronounced acute arousal symptoms were (m=9.55 days after hospital admission) also those who showed the least symptom improvement across a 12 month period . Thus, the role of arousal appears pronounced in both early and long term posttraumatic symptomatology . Thus, it is also pertinent to differentiate between different forms of arousal both in relation to asd and ptsd . Given that the proposed dysphoric arousal model separates the original hyperarousal factor, it is important to investigate the inter - factor correlations . With this in mind, the current study found a certain degree of orthogonality between the dysphoric arousal and anxious arousal factors (r=.49) of the asd dysphoric arousal model . Indeed, this finding is contrary to that of some ptsd factor analytic studies, which have previously (but not always) reported high inter - factor correlations between dysphoric arousal and anxious arousal (armour et al ., 2012). However, a moderate correlation between these two factors adds strength to the rationale behind their separation (cf . Interestingly, separating the original arousal factor into dysphoric arousal and anxious arousal resulted in a non - significant correlation between the latter and avoidance . One possible explanation for this is that avoidant coping may theoretically and practically overlap with the ptsd avoidance factor, in that avoidance symptoms may act as coping processes for alternative posttraumatic symptomatology (as argued by leiner, kearns, jackson, astin & rothbaum, 2012). Due to the close similarity between the asd and the ptsd avoidance items and factor, this overlap is likely also apparent between asd avoidance symptoms and avoidant coping behaviors . Thus, the lack of a significant correlation between avoidance and anxious arousal in the current study may indicate that avoidance has functioned as a coping mechanism and thus has prevented the development of anxious arousal . Indeed, the potentially protective function of the avoidance symptoms may also be indicated by the low to moderate correlations between the avoidance factor and the remaining asd factors in the present study . In accordance with the present study, leiner et al . (2012) also found that avoidant coping was negatively associated with ptsd severity in treatment seeking rape victims . Furthermore, leiner et al . (2012) found that the association between avoidant coping and ptsd severity was still significant after controlling for initial ptsd severity and after the removal of ptsd avoidance symptoms in order to account for the possible overlap between avoidant coping and ptsd avoidance symptoms . Although, the majority of the sample in present study were rape victims, avoidant coping has also previously been found protective against posttraumatic symptoms following robbery (elklit, 2002). In contrast, however, holahan and moos (1987) reported that although avoidant emotional coping may be adaptive in the immediate aftermath of trauma, such becomes maladaptive if relied on over a prolonged period of time (see also olff, langeland & gersons, 2005). Indeed, schnider, elhai and gray (2007) concluded that avoidant coping is one of the best predictors of ptsd severity in college student who had suffered a traumatic loss . Notably however, the non - significant correlation is not with hyperarousal specifically, but with a relatively new grouping of symptoms; anxious arousal . This particular constellation of symptoms is based on fear response rather than anxiety per se, thus the lack of association between avoidance and anxious arousal is, as of yet, unexplored territory . In contrast to the majority of the previous cfa studies (brooks et al ., 2008; hansen et al . The dsm - iv model was not supported in the present study and neither was the armour et al . As mentioned earlier, it is possible that the discrepancy in the previous cfa studies of asd is caused by variation in the form of traumatic exposure or the asd prevalence rates as recently found in relation to ptsd (biehn et al ., 2012). Both are apparent in relation to asd, because the dsm - iv model was found to be the best fit following bank robbery (hansen et al ., in press) in a sample of all participants exposed to the trauma, whereas the three - factor model was found to be the best fit following rape (armour et al ., 2011) in a sample were only those participants who met the diagnostic criteria for all four asd symptom clusters was assessed . Notably, however the dysphoric arousal model was not tested in previous studies and thus may have provided superior fit in the varying trauma and asd prevalence samples . Thus, taking these points and the current results into consideration, the separation of the arousal factor into separate dysphoric and anxious arousal factors may point to an asd model which represents asd's underlying dimensionality across heterogeneous trauma events better than that of the previous models . The current study is the third (apart from armour et al ., 2011; hansen et al ., in press) to investigate the fit of the uni - dimensional, one - factor model proposed for the dsm-5 . Notably, none of the studies, including this one, have found support for this model over and above alternative models . Thus, despite certain discrepancies in factor analytic results related to asd's latent structure, and despite the only recent (thus, not fully explored) proposal that the dysphoric arousal model may represent asd's latent structure, it is becoming increasingly clear that the current proposal of asd in the dsm-5 seems to be an unsuitable description of acute traumatic stress symptoms . In other words, a growing body of empirical literature highlights the need for accommodating the diagnostic criteria in the dsm-5 . Indeed, not doing so may have a serious negative impact, given that assessment and treatment may be based on an imprecise theoretical model . On a more promising note, bryant et al . (2011) proposed that the inclusion of a wider range of acute reactions in asd in the dsm-5 would result in a more precise conceptualization of asd than previously . However, alternative proposals of asd in the dsm-5 need to be put forward so that they can be tested . Limitations of the present study include using a self - report measurement of asd rather than a clinician - administered interview . Thus, it is possible that the results reflect properties of the asds rather than the asd diagnosis per se . However, this does seem unlikely, given that the asds items are specifically matched to each of the asd symptoms and given that, unlike previous studies, we used a 100% clinical sample (i.e., all participants met diagnostic criteria). However, as highlighted by brooks et al . (2008) it is important to underline that the results should not be interpreted in relation to the utility of the asd diagnosis . It is also important to note that the generalization of these results to alternative trauma populations must be conducted with caution . This is attributable to the fact that although we used a heterogeneous sample of bank robbery and rape victims, the latent structure may vary in trauma populations which have not experienced interpersonal assault . It is also important to note that the large majority of participants in the current study were female, thus the results may not generalize well to male populations, given that gender may also moderate asd's latent structure . Furthermore, we did not assess the individual factors of the asd dysphoric arousal model in relation to external psychopathological constructs . Armour et al ., 2012) and as such would be an interesting line of enquiry in relation to the asd dysphoric arousal model . Moreover, albeit that the dysphoric arousal model was the optimal model compared to alternatives in the current study, model fit was not ideal with excellent model fit only being indicted by the rmsea value and corresponding confidence intervals . In spite of these limitations, the present study represents the first examination of the latent structure of asd in a heterogeneous clinical assault sample . In addition, the present study is based on the largest asd clinical sample thus far . Moreover, the present study is the first study to examine a replication of the five - factor dysphoric arousal model of ptsd which separates the arousal factor into dysphoric arousal and anxious arousal factors . The results of the present study suggest that the dimensionality of asd may best be represented as a five factor structure following exposure to two heterogeneous assault types . Thus, the current study adds to the debate about how asd should be conceptualized in the pending dsm-5 . Future research on asd's latent structure should further test the dysphoric arousal model across varying forms of traumatic experience . Future research may also wish to assess if the asd dysphoric arousal model remains invariant across sub - populations (i.e., split by gender, culture, and perhaps diagnostic status). There is no conflict of interest in the present study for any of the authors.
Gierke, in 1905, described a lesion of adrenal gland containing fat and myeloid elements . A 40-year - old man referred to department of endocrinology with adrenal mass and hypertension . He was diagnosed with hypertension 3 years back, initial bp was 180/110 mm hg, was started on anti - hypertensive treatment . On examination, there were no neurocutaneous markers or marfanoid habitus, no features of cushing's syndrome, 24-hour urine metanephrines level was 3000 micrograms / day (normal <900 micrograms / day, the test was done after stopping all interfering drugs). Ultrasonography revealed 9.8 8.5 cms well - defined predominantly hyperechoic lesion, faint hypoechogenicity originating from right suprarenal region abutting the upper pole of right kidney and lower surface of right lobe of liver suggestive of right adrenal mass . Cect of abdomen showed 9.8 8.5 cm well - defined, well - circumscribed heterogenous hypoattenuated mass lesion noted in right suprarenal region and minimal enhancement on contrast with 80 to 100 hf units of attenuation suggestive of myelolipoma of right adrenal gland [figure 1]. Abdominal contrasted computerized tomography showing well - defined non - homogeneous mass of right adrenal origin in view of hypertension, adrenal mass, and elevated 24-hour urine metanephrines (> 3 times), possibility of pheochromocytoma was considered . Patient underwent surgery, and well encapsulated right adrenal tumor (weight: 500 gm) was excised [figure 2]. Immuno - histochemistry of specimen revealed positive for chromogranin a, suggestive of catecholamine - secreting granules in the tissue [figure 4]. Gross specimen of removed mass h and e staining revealing features of myelolipoma with mature fat cells, suspended with plenty of normal hematopoitic marrow elements with congested blood vessels . (original magnification, 100) immuno - histochemistry of specimen revealed positive for chromogranin a the patient had remission in hypertension . Adrenal myelolipomas are uncommon benign tumors of adrenals, composed of adipose and hematopoietic tissue in varying proportions, a result of metaplasia of reticuloendothelial cells . As these tumors are usually more than 5 cms in diameter, they can be easily detected on ultrasound . The lesion is typically seen as a well - encapsulated heterogeneous supra - renal mass of low density with negative attenuation values, interspersed by dense myeloid tissue and with or without specks of calcification . The diagnosis of adrenal myelolipoma in our case was suggested by the established ct scan criteria [figure 1] and the typical histopathological features [figure 3]. Functionality of the adrenal mass in this patient was suggested by the presence of hypertension and elevated metanephrines . Catecholamine secreting adrenal myelolipoma was confirmed by the absence of any evidence of pheochromocytoma on hpe and documentation of positive staining for chromogranin a on ihc [figure 4]. Brogna et al ., reported a giant cortisol secreting adrenal myelolipoma, but our patient was clinically and biochemically eucortisolic . To the best of our knowledge, only two case reports are available on catecholamine - secreting adrenal myelolipoma in the world literature . Tamidari et al . Have reported a case of a large, right - sided catecholamine, secreting adrenal myelolipoma with increased 24 hours urinary metanephrines . Udupa et al . Have reported a large adrenal myelolipoma with increased 24 hours urinary vanillylmandelic acid (vma) levels . All these patients became normotensive and biochemical abnormalities normalized following surgery similar to the patient in our case . The association of adrenal myelolipoma and hypertension may not be entirely coincidental, as it may be associated with catecholamine secretion, as seen in our case.
Diabetes mellitus is a chronic metabolic disorder that is distinguished by hyperglycemia, underutilization of blood glucose, and defects in the metabolism of macronutrients such as carbohydrates, fat, and protein, secondary to the disturbance in insulin action [1, 2]. Type 1 diabetes is caused as a result of insulin deficiency due to failure of pancreatic beta - cells . Therefore, diabetic patients need exogenous insulin injection, whereas patients with type 2, known as insulin independent, do not respond to insulin and consequently can be managed by lifestyle changes . Next to cancer and heart disease, diabetes mellitus is the third most life - threatening disorder which has a high rate of morbidity and mortality worldwide . Prevalence of this disorder is quickly rising in every part of the world . As a result, it is projected to affect 552 million people in 2030, rising sharply from 336 million in 2011 . Signs of type 1 and 2 diabetes may include hyperglycemia, abnormal thirst, repeated urination, weight loss and severe hunger, nausea and vomiting, mood changes, and severe weakness and tiredness . The purpose of this review was to identify anethum graveolens l. (ag) as a valuable herbal plant, originating from asian traditional medicine, in the management of diabetes . Conducted studies could allow representing the potential applications for future research . To complete the assumed task, traditional sources regarding the usage and the carried out investigation on anethum graveolens l. are collected and explained . Essential assessments of the documented bioactive components particularly in terms of their correlation with recognized traditional use and current pharmacological knowledge are discussed . Phytochemical studies, antidiabetic properties, suggested mechanism, and the main compounds of ag that may be responsible for useful effects of ag are described too . To prepare this review a pharmacological and phytochemical literature survey was performed using scopus, pubmed, and web of science . Studies have shown that blood lipid peroxidation markedly rises in diabetes due to vascular injury, which is provoked by hyperglycemia and free radicals . This peroxidation can further aggravate the progress of diabetic complications and is thought to happen as a result of defects in insulin secretion by pancreatic -cells . Diabetes also leads to an impaired lipid profile, including low levels of high - density lipoprotein (hdl) cholesterol and hypertriglyceridemia and raised oxidative stress [30, 31]. Cvd is the most common complication in diabetes, which is a common reason for premature mortality in diabetic patients . As a result, to reduce the risk of cvd, many studies potentially recommended control of dyslipidemia and hyperglycemia [1, 3234]. In addition to dyslipidemia, hyperglycemia is known as a major risk factor for diabetic complications . Protein glycation is involved in the progress of complications such as retinopathy, neuropathy, nephropathy, dermopathy, and atherosclerotic lesions [1, 33, 3537]. In the chronic state, excess glucose binds to free amino groups in tissue or blood proteins and body fluid and disturbs their functions [3840]. Age formation is known as a key element in diabetes complications [4146]. In diabetic patients, reactive substances such as hydroxyl radicals (oh), hydrogen peroxide (h2o2), lipid peroxyl radicals (loo), superoxide anion (o2), and peroxyl radicals (roo) the reaction of these reactive species with polyunsaturated fatty acids (pufas) causes lipid peroxidation which is consequently implicated in early diabetes complications, especially premature cardiovascular disease [47, 48]. For instance, superoxide dismutase (sod) is known as one of the main endogenous enzymes that removes o2 and produces h2o2, which is then processed to water by the activity of catalase (cat) and glutathione peroxidase (gpx). Hence, sod can work as the chief protector against o2 and it inhibits production of free radicals . Cat is an important enzyme involved in detoxification of hydrogen peroxide to water and oxygen . On the other hand, glutathione s - transferase (gst) and glutathione peroxidase (gpx) gpx decreases free radical damage by preventing the production of extremely cytotoxic species, for instance, lipid peroxides and other organic hydroperoxides . Gst is therefore believed to preserve the balance of cellular redox conditions [47, 49]. Lipid peroxidation naturally occurs on a small level in the body and plays a key role in diabetes and cardiovascular diseases pathogenesis . The reactive oxygen species such as hydroxyl radical and hydrogen peroxide attacks polyunsaturated fatty acids and causes lipid peroxidation . Iron ion and hydrogen peroxide in a known process, called fenton reaction, lead to formation of hydroxyl radical which is a potent oxidative agent . Oral administration of many herbal plants leads to a marked rise in the activities of sod, cat, gsh, gst, and gpx in diabetes [51, 52]. There are different ways for treatment and control of diabetes, including administration of insulin or hypoglycemic drugs, dietary control, and exercise [53, 54]. Furthermore, chemical drugs generally have low efficacy over time, are too expensive, and show severe side effects such that some patients cannot tolerate prolonged treatment or taking the high dosages of these drugs . In contrast, herbal medicines are regarded as potential sources for medication due to their useful effects, negligible side effects in clinical trials, and low cost [5359]. People use different medicines such as sulfonylureas (glimepiride, glipizide, and glyburide), biguanides (metformin), thiazolidinediones (pioglitazone), alpha - glucosidase inhibitors (acarbose), meglitinides (nateglinide), and dipeptidyl peptidase 4 inhibitors (januvia, onglyza) for control and treatment of hyperglycemia . More than 10 to 20 percent of diabetic patients are known to eventually stop taking their medications because of the side effects . Some of the diabetes medicines can also cause hypoglycemia which is a harmful and potentially lethal side effect . Additional side effects of several antidiabetic drugs are weight gain, increased risk for fractures especially in women, and gastrointestinal disorders such as abdominal pain, gassiness, vomiting, nausea, diarrhea, and bloating . Edema and increased level of low - density lipoprotein cholesterol (ldl - c) were also reported by many patients . Uncommon side effects include congestive heart failure, allergic reactions, and anemia, which can be induced by chemical diabetic medicines [30, 31]. Nowadays, many physicians prescribe insulin, thiazolidinediones, sulphonylureas, -glucosidase inhibitors, and so forth, for treatment and control of diabetic complications . Nevertheless, since adverse effects of these medicines are arguable, there is a need for novel agents in diabetes treatment . In this respect, some herbal medicines due to their lower side effects, low costs, useful properties such as antihyperglycemic, antihyperlipidemic, and antioxidant effects, and especially their natural origin have been proposed for potential management of diabetes [76, 77]. However, because of unknown mechanisms, only a small number of plants have been used for the management of diabetic complications . Herbal plants contain many chemical components such as flavonoids (30%), terpenoids (17%), saponins (9%), polyphenols (6%), alkaloids (5%), tannins (4%), polysaccharides (3%), and miscellaneous compounds (17%), which are known to have remedial properties . Interestingly, the world health organization (who) reported that about 80% of the world's people presently use herbal plants for treatment of different diseases . Although many plants have been suggested for treatment of diabetes due to their antidiabetic properties, in this review, we will attempt to open a new window toward application of ag and its constituents in the management of type 2 diabetes . Anethon and the colloquial name of dill is derived from the old norse word ag is the only species of the genus anethum, still classified by several botanists related to peucedanum genus as peucedanum graveolens (l.). Ag is an annual vertical plant with 50150 cm stem belonging to the apiaceae family . The fruits of ag are brown, flat, tiny, and oval in shape (figure 1). Ag contains 36% carbohydrates, 15.68% proteins, 14.80% fiber, 9.8% ash, and 8.39% moisture as well as essential oils, fatty oil, minerals, and vitamins . Ag is used in the traditional herbal medicine for the management and prevention of digestive disease, breath problem, motivation of lactation, and also reduction of cholesterol and glucose . Currently, many studies have established these properties; also, ag is recently known as anticancer, antimicrobial, antigastric irritation, anti - inflammatory, and antioxidant agent . In this respect, dill is produced as a hypolipidemic drug (anethum tablet) in iran which consists of anethum graveolens (68%), cichorium intybus (5%), fumaria parviflora (5%), and lime (citrus aurantifolia) (4%). Administration of different extractions of ag seed and leaf, as well as its essential oil, in diabetic models significantly reduced triglycerides, total cholesterol, low - density lipoprotein cholesterol (ldl - c), very - low - density lipoprotein cholesterol (vldl - c), and glucose levels, whereas it increased hdl - c level . On the other hand it has also been shown that the extract from ag flowers had more antioxidant activity than either its seed or leaf extract . Duration of treatment in different experiments was variable; for example, koppula and choi used only single dose for one hour, while the treatment period in mansouri et al . However, almost all of the experiments show that one month is an appropriate time for survey of antidiabetic properties of dill . To survey the effects of ag on dyslipidemia the rabbit is known as an animal model that has been commonly used for the study of hypercholesterolemia . Only few studies have been done on rabbits, whereas rats are an optional model for diabetes studies . Therefore, as mentioned in table 1, most of the studies used diabetic rats . Inhibition of intestinal cholesterol absorption, binding to bile acids in the intestine, an increase in fecal excretion, and increasing production of bile acids might be the main mechanisms by which ag exerts its antidiabetic functions . [24, 82] proposed that some components of ag such as carvone, limonene, or -phellandrene are responsible for the hypolipidemic properties of ag, likely through reducing acyl coa carboxylase or 3-hydroxy-3-methylglutaryl - coa (hmg - coa) reductase, the key enzymes in fatty acid and cholesterol metabolism . Other studies have suggested that ag components might rise liver ldl receptors, decrease fatty acid synthesis, and impact lipoprotein homeostasis mainly through improvement in lipoprotein metabolism . Furthermore, some studies suggest that ag may improve the lipid profile by its antioxidant properties . We have also observed that administration of 100 and 200 mg / kg of ag tablet and hydroalcoholic extract of ag in hypercholesterolemic hamsters resulted in significantly reduced hmg - coa reductase activity and mrna levels . For the first time, we also showed that ag normalized the lipid profile by decreasing hmg - coa reductase in the hypercholesterolemic hamsters and type 2 diabetic rats . We have also shown that ag increased ldl receptor level in the liver resulting in stimulation of cholesterol clearance from the bloodstream (unpublished data). The results of abbasi oshaghi et al . 's study showed that ag significantly increased cholesterol-7-alpha hydroxylase activity, the rate - limiting enzyme in bile acids biosynthesis . Some studies have also reported that rutin and quercetin (components of dill) may decrease serum cholesterol and ldl - c levels and decrease liver cholesterol level . . Showed that treatment of diabetic rats with different extracts of ag normalized lipid deposits in the liver, pancreas, and heart . Hplc analysis showed that ag has high amounts of quercetin which may be responsible for the suppression of hmg - coa reductase activity . Numerous studies that have previously shown the antidiabetic effects of ag are listed in table 1 . Chemical compounds of ag extracts especially flavonoids, terpenoids, alkaloids, tannins, and phytosterols are probably responsible for its hypoglycemic effects . We found that different extracts of ag reduce ages formation [85, 86]. Our study also showed that ag at the dose of 300 mg / kg had potential antioxidant and hypolipidemic effects . Reported significant decline in fasting blood glucose level with 300 mg / kg hydroalcoholic extract of anethum graveolens . Doses of ag up to 2 g / kg in a short period of time did not cause any death in animal models . Also, the ag doses up to 1000 mg / kg did not cause any toxic effect in short time experiments . Although different doses of ag were used in animal models, ahmadi mahmoudabadi reported that the effective dose for metabolic properties is 300 mg / kg in animal models . Different studies showed that both high and low doses of ag have hypoglycemic and hypolipidemic effects . The doses less than 50 mg / kg could be known without adverse effect in rats and mice . However, piri et al . Demonstrated that 3-week administration of anethum extract at the doses of 50, 100, and 200 mg / kg normalized blood lipid, and the change in blood glucose was not significant . The safe and effective dose in human studies is reported as 650 mg / kg . In an in vitro study, abbasi oshaghi et al . Found that different extracts of ag at the concentrations of 0.032, 0.065, 0.125, 0.25, 0.5, and 1 mg / ml reduced ages formation; fructosamine level and carbonyl groups, also, raised thiol groups in type 2 diabetic animals . Oxidation of carbonyl and thiol groups on blood proteins significantly reduces by anethum in a dose dependent manner . According to the study conducted by adisakwattana et al ., polyphenolic compounds in plant extract may play a major role in the inhibition of ages formation . Ages are recognized to contribute to the production of oxidative stress and inflammation, which are related to the diabetes complications . It has been reported that administration of age - rich diets in an animal model is associated with rising of blood and tissue ages and the conditions like atherosclerosis . . Showed that administration of ag powder in type 2 diabetic patients reduced fasting blood glucose and normalized insulin resistance and lipid profiles . As mentioned above, ag is rich in antioxidant compounds; therefore, antioxidant and flavonoid components of ag probably are able to repair damaged -cells and insulin secretion . It has been shown that ag significantly increases total antioxidants in the pancreas and also regenerates histopathological changes . Takahashi et al . Have shown that ag extract normalized lipid profile in diabetic obese mice by activating peroxisome proliferator - activated receptor- (ppar-) and increasing fatty acid oxidation - related genes expression . Our colorimetric and hplc analysis also showed that dill had a high amount of quercetin, phenol, flavonoid, tannin, saponin, and alkaloid . Therefore, we can conclude that components of dill are responsible for its metabolic effects . Mishra reported that aqueous extract of ag has a high amount of carvone, which is accountable for its antidiabetic activity . The ethanolic extract of ag which is prepared in india showed very low antidiabetic properties . On the other hand, mudassir et al . Reported that the prepared ethanolic extract of ag in pakistan significantly reduced blood glucose (34% reduction). They declared that hypoglycemic effects of ag might be due to the inhibition of intestinal glucose absorption . In our laboratory, we have previously shown that ag has potential antioxidant activity . Our results are in agreement with the results of a previous study by bahramikia and yazdanparast, indicating dpph radical scavenging activity of ag . In our study, 1 mg / ml of ag water extract showed 96.04% scavenging of h2o2, whereas only 27.26% radical scavenging activity was reported for ag ethanolic extract . Ag also showed fe ion chelation which is known as a defense mechanism against oxidative damage by declining free radical production via fenton reaction . In this reaction, iron(ii) is oxidized by h2o2 to create potential free radicals such as hydroxyl radical . The results of a study by bahramikia and yazdanparast showed that crude extract of ag exhibited a moderate no scavenging activity . In line with this observation, we showed a nitric oxide scavenging activity of ag similar to the known radical scavengers, ascorbic acid and butylated hydroxytoluene (bht). Interestingly, we showed that no scavenging activity of ag is comparable with the activity of ascorbic acid and/or bht . We showed that a 1 mg / ml solution of dill water extract has 90.41% no scavenging activity which was much greater than that observed (78.49%) for ethanolic extract of ag by orhan et al . . We have also detected a greater antioxidant activity for ag water extract compared with methanolic extracts . Interestingly, the antiradical and antioxidant properties of ag in all tests were similar to ascorbic acid and bht . The results of many studies established the notion that the particular parts of dill are often discriminated by significant diversity in their constituents' chemical compounds . It has been reported that ethanolic extract of ag has very low antidiabetic properties when compared with the aqueous extract concordant with the hypoglycemic function of ag ethanolic extract, other studies have reported that this extract significantly reduces blood lipids [24, 82]. Interestingly, we showed that different extracts of ag significantly reduced glucose level, but the reduction by water or hydroalcoholic extract was more as compared with the methanolic extract . Furthermore, with review of the literature, we can find that different preparations such as hexane, ethanolic, water, and hydroalcoholic extracts of seed and leaf have been used in different studies (table 1). There is some diversity in the dill composition in different countries; for example, american dill oil has a high amount of alpha - phellandrene, while carvone and limonene are the main constituents of asian and european dill . In addition to essential oils especially carvone, limonene, and alpha - phellandrene, dill also contains fatty oil, proteins, carbohydrates, moisture, fiber, ash, vitamins (a and niacin), and mineral elements (calcium, potassium, magnesium, phosphorus, and sodium). This plant is known as a rich source of flavonoids, phenolic compounds, saponins, cardiac glycosides, and terpenes . Jana and shekhawat reported that there are several volatile components of ag seeds and herb . For instance, carvone is the main odorant of ag seed and limonene, -phellandrene, myristicin, and dill ether are the most vital odorants of ag herb . Larijani et al . Also reported that seeds and leaves of ag have different components and consequently have different therapeutic effects . Different parameters such as cultivating area, genotype, and environmental factors have huge effects on the composition of ag extracts . Furthermore, reichert et al . Reported that the different parts of ag have different compounds and these compounds may change depending on the stage of plant growth . For example, sefidkon reported that -phellandrene and limonene are the major constituents of dill oil, while singh showed that carvone, dill apiole, limonene, trans- and cis - dihydrocarvone, and linalool are the main constituents . Consequently, the different components which are reported by the different studies also depend on the different plant growth stage, cultivating area, and the particular part of the plant that is used . Different compounds of ag have been described in the different published reports which are listed in table 2 . However, it has been shown that ag administration is safe, and it may rarely lead to allergic reactions, vomiting, diarrhea, oral pruritus, urticaria tongue, and throat swelling; furthermore, it is not suggested in pregnancy . Mirhosseini et al . Reported that ag did not have any side effects, as compared with gemfibrozil, in treated patients . Other clinical trial studies did not report the side effect of ag [11, 13, 26, 27]. Herbal medicines have multiple key components such as flavonoids, terpenoids, saponins, polyphenols, tannins, alkaloids, and polysaccharides that have their individual remedial properties . Herbal medicines especially ag have a lot of useful properties including antihyperlipidemic, antihypercholesterolemic, antidiabetic, anticancer, antioxidant, antistress, antisecretory, cardioprotective, antispasmodic, and diuretic effects . Moreover, epidemiologic experiments reported a converse relationship between ag consumption and risk of diabetes and cvd progression . Recent literature strongly supports the suggestion that consumption of ag has a significant antidiabetic effect in both humans and animals . According to the reported antidiabetic effects of dill, it can be suggested for the management of diabetic patients . However, the diverse preparations, dose of ag, period of ag consumption, and interaction with other drugs must be normalized . More studies are required to recognize the specific constituents of ag that are accountable for most of its valuable properties . Although all components of ag have antioxidant, hypoglycemic, and hypolipidemic effects, these components are at low levels in dill and possibly have synergic effects . Moreover, the findings suggest that activation of ldl - r, ppar - alpha, and other fa oxidation - related genes and also inhibition of hmg - coa reductase contribute to the hypolipidemic effects of ag.
Male homozygous db / db and age- or sex - matched heterozygous littermates db/+ were obtained from the jackson laboratory (bar harbor, mi). Male balb / c mice were purchased from charles river laboratories (wilmington, ma). Mice were housed in standard shoebox cages supplied with standard rat diet and water ad libitum in a pathogen - free facility with a 12-h light / dark cycle . For lps treatment, male balb / c mice (25 g) were injected intraperitoneally with lps at the dose of 4 mg / kg body weight for 2 consecutive days, as described (35). To isolate peritoneal macrophages, mice were killed and peritoneal lavage fluid was collected by washing the peritoneum with 5-ml ice - cold hank's balanced salt solution (hbss; gibco, carlsbad, ca). Subsequent preparation of peritoneal macrophages was according to the protocol as described (35). All procedures were approved by the institutional animal care and usage committee of children's hospital of pittsburgh . Other methods including the statistics are described in the supplemental materials available in an online appendix at http://diabetes.diabetesjournals.org/cgi/full/db09-0232/dc1 . Aberrant proinflammatory cytokine production is intertwined with insulin resistance in obesity and type 2 diabetes . To decipher the molecular basis that links proinflammatory cytokine production to insulin resistance, we determined the effect of foxo1 on cytokine production in macrophages, a cell type that plays a pivotal role in mediating whole - body insulin resistance in the face of low - grade inflammation . Macrophage raw264.7 cells were transduced with adv - foxo1 or adv - null vector at a predefined moi (200 pfu / cell), followed by the determination of cytokine concentrations in conditioned medium . As shown in fig . 1, transduction of raw264.7 cells by foxo1 vector resulted in a significant induction of il-1 and il-2 production without alterations in the production of other cytokines including il-4, il-5, il-6, il-12, ifn-, tnf-, and granulocyte monocyte colony - stimulating factor . In contrast, il-10 expression was significantly downregulated in response to foxo1 production in raw264.7 cells . In the absence of lps, the expression of proinflammatory cytokines remained at low basal levels independently of foxo1 action (supplemental table s1). These data underscore the importance of foxo1 in regulating cytokine production in lps - activated macrophages . Because of the close association between abnormal production of il-1 in macrophages and the pathogenesis of insulin resistance, we focused on the delineation of the molecular basis underlying foxo1-mediated induction of il-1 in this study . Raw264.7 cells were mock - treated with pbs, or transduced with adv - foxo1 or control adv - null vector at a fixed dose (moi, 200 pfu / cell). After 24-h incubation in the presence of 100 ng / ml lps, conditioned medium was harvested for profiling the production of cytokine il-1 (a), il-2 (b), il-4 (c), il-5 (d), il-6 (e), il-10 (f), il-12 (g), ifn- (h), tnf- (i), and granulocyte monocyte colony - stimulating factor (gm - csf) (j). * p <0.001 vs. controls . To investigate the potential role of foxo1 in macrophage production of il-1 in vivo, we treated balb / c mice with once - daily intraperitoneal administration of lps (4 mg / kg) for 2 days . Compared to pbs - treated control mice (n = 8), lps - treated mice (n = 8) exhibited significantly elevated plasma il-1 levels (fig . Two days after lps treatment, mice were killed and peritoneal macrophages were retrieved for real - time qrt - pcr analysis . 2b), correlating with significantly elevated il-1 mrna levels in peritoneal macrophages (fig . Male balb / c mice (8 weeks old, n = 8 per group) were treated with once - daily intraperitoneal injection of lps (4 mg / kg body weight) or mock - treated with pbs for 2 days . Aliquots of blood (50 l) were sampled from the tail vein for the determination of plasma il-1 levels (a). Two days after lps treatment, peritoneal macrophages were isolated from individual mice for the preparation of total rna, which was subjected to real - time qrt - pcr assay for the determination of foxo1 (b) and il-1 (c) mrna levels using 18s rrna as control . * p <0.05 and * * p <0.001 vs. control . To determine whether foxo1 plays a role in linking insulin resistance to aberrant il-1 expression, we studied foxo1 expression in peritoneal macrophages in obese db / db mice . Because of the lack of leptin receptor, db / db mice are genetically obese, developing insulin resistance and hyperinsulinemia at weaning . Two groups of male db / db mice (n = 7, male, 10 weeks old) and age- or sex - matched heterozygous db/+ lean littermates (n = 7) were fasted for 16 h, as described (30). Peritoneal macrophages were isolated from individual mice and subjected to real - time qrt - pcr analysis . As shown in fig . 3a, foxo1 expression was significantly upregulated in peritoneal macrophages of insulin - resistant obese db / db mice . Furthermore, db / db mice were associated with significantly higher levels of il-1 mrna in peritoneal macrophages (fig . 3c), which was indicative of chronic inflammation in insulin - resistant obese db / db mice . Covariation of foxo1 and il-1 expression in peritoneal macrophages of insulin - resistant db / db mice . Peritoneal macrophages were isolated from insulin - resistant obese db / db mice (n = 7) and control db/+ littermates (n = 7). Total macrophage rna was prepared and subjected to real - time qrt - pcr assay for the determination of foxo1 (a) and il-1 (b) mrna levels . We hypothesized that the il-1 gene is a foxo1 target, and foxo1 promotes il-1 expression in macrophages in response to inflammation . To test this hypothesis, we treated raw264.7 cells with palmitate, the predominant saturated form of free fatty acid that is known to elicit low - grade inflammation . 4, this treatment resulted in significant induction of il-1 production, as reflected in markedly elevated levels of il-1 protein in conditioned medium and il-1 mrna in cells . / l palmitate or bsa as control for 24 h. cells were harvested and subjected to real - time qrt - pcr assay for the determination of foxo1 (a) and il-1 (b) mrna levels . A 2-kb dna fragment harboring the mouse il-1 promoter was cloned into the pgl3-basic luciferase reporter system (fig . Il-1 promoter activity was determined in response to adenovirus - mediated foxo1 production in the presence and absence of insulin . As shown in fig . 5b, we detected a more than sixfold induction of the il-1 promoter activity in raw264.7 cells that were pretransduced with foxo1 vector . This effect was counteracted by insulin, correlating with the ability of foxo1 to undergo insulin - dependent translocation from the nucleus to cytoplasm (25,27). A: a schematic depiction of the il-1 promoter directed luciferase reporter system in phd386 . Raw264.7 cells in six - well microplates were transduced with adv - foxo1 or control adv - null vectors at a fixed dose (moi, 200 pfu / cell), followed by transfection with 1-g phd386 plus 1-g pca35-lacz in the presence of 100 ng / ml lps in culture medium . After 24-h incubation in the absence or presence of insulin (300 nmol / l), cells were harvested for the determination of luciferase and -galactosidase activities . The relative luciferase activity was calculated by setting the ratio of luciferase/-galactosidase activity in control cells to 1 . Raw264.7 cells were transfected with wild - type (wt) or mutant (mt) il-1 promoter directed luciferase system in the absence or presence of adenovirus - mediated foxo1 production . After 24-h incubation in the presence of 100 ng / ml lps, cells were harvested for the determination of relative luciferase activities using -galactosidase activity as internal controls . Raw264.7 cells were cotransfected with phd386 and pca35-lacz in the absence or presence of adenovirus - mediated foxo1 production . One subset of cells was transduced with 200 moi of adv - foxo1-rnai encoding foxo1-specific rnai or control adv - scrnai encoding scrambled rnai . After 24-h incubation in the presence of 100 ng / ml lps, cells were harvested for the determination of relative luciferase activities using -galactosidase activity as internal controls . The conditioned medium from the experiment in d was used for the determination of il-1 levels . Cells obtained from the experiment in d were subjected to semiquantitative immunoblot analysis for the determination of total foxo1 protein levels, using intracellular actin protein as control . Raw264.7 cells incubated in six - well dishes in the presence of lps (100 ng / ml) for 24 h. cells were fixed with 1% formaldehyde, followed by chip assay using rabbit anti - foxo1 or control igg (rabbit anti-galactosidase). Immunoprecipitates were analyzed by pcr assay for the detection of dna, using primers flanking the consensus foxo1 binding site within the il-1 promoter . Ns, not significant . To corroborate these findings, we altered the insulin - responsive element (ire) motif within the il-1 promoter via site - directed mutagenesis (supplemental fig . The resulting mutant il-1 promoter was analyzed for its ability to mediate the stimulatory effect of foxo1 on il-1 expression . 5c, alterations within the ire dna motif rendered the mutant il-1 promoter unresponsive to foxo1 production in raw264.7 cells . As control, adenovirus - mediated foxo1 production resulted in a significant induction of wild - type il-1 promoter activity (fig . 5c). To further prove that foxo1 contributed to il-1 regulation, we employed an rna interference (rnai)-mediated gene silencing approach to knockdown foxo1 expression in cultured macrophages . Raw264.7 cells were transfected with phd386 expressing the il-1 promoter - directed luciferase reporter gene, followed by transduction of adenoviral vectors expressing foxo1-specific rnai or control scrambled rnai, as described (30). After 24-h incubation, cells were subjected to luciferase activity assay . As shown in fig . 5d, rnai - mediated foxo1 knockdown resulted in abolition of foxo1-mediated induction of il- promoter activity in lps - stimulated raw264.7 cells . These results were corroborated when the conditioned medium was used for the determination of il-1 protein . This effect was abolished by rnai - mediated foxo1 knockdown, as reflected in 80% of reduction in foxo1 protein levels in raw264.7 cells that were pretransduced with foxo1-rnai vector (fig . The above results spurred the idea that foxo1 stimulates il-1 expression via direct binding to the il-1 promoter . 5a), a consensus dna motif that is responsible for binding and mediating foxo1 induction of target gene expression (25,27). Interestingly, similar foxo1 consensus sites were detected in the regulatory region of both human and rat il-1 promoters, suggesting an evolutionally conserved mechanism (supplemental fig . S2). To determine the ability of the conserved ire motif to mediate the stimulatory effect of foxo1 on il-1 promoter activity, we performed chromatin immunoprecipitation (chip) assay to analyze the potential interaction of foxo1 with il-1 promoter dna in raw264.7 cells . Because of low foxo1 expression in unstimulated raw264.7 cells, we transduced raw264.7 cells with foxo1 vector in the presence of lps (100 ng / ml) in culture medium . After 24-h incubation, cells were subjected to chip assay using rabbit anti - foxo1 antibody or control rabbit igg . The resulting immunoprecipitates were subjected to pcr analysis, using primers flanking the ire motif within the il-1 promoter . A 457-bp dna corresponding to the ire region (1,359/902 nt) of the il-1 promoter was produced from anti - foxo1 immunoprecipitates (fig . 5f). In contrast, the immunoprecipitates derived from control igg were negative in the same pcr assay . To further illustrate the role of foxo1 in linking aberrant il-1 production to insulin resistance, we determined the effect of lps or palmitate treatment on insulin signaling in cultured macrophages . Raw264.7 cells were incubated in the presence of lps (100 ng / ml) or palmitate (0.2 mmol / l) for 36 h, followed by the addition of insulin (300 nmol / l) into culture medium . The dose of insulin used in culture medium was based on our preliminary studies and data in the literature (36). Cells were harvested 45 min after insulin action for the determination of insulin - stimulated phosphorylation of insulin receptor substrates (irss) and foxo1, both of which are key signaling molecules downstream of insulin action . Compared to mock - treated controls, raw264.7 cells pretreated with lps or palmitate treatment were associated with a significant reduction in insulin - stimulated phosphorylation of irs2 (fig . Because of extremely low levels of irs1 expression in raw264.7 cells, we were unable to detect insulin - stimulated phosphorylation of irs1 . (37), who show that irs1 is undetectable and irs2 is the sole substrate downstream of insulin action in primary murine macrophages . Lps or palmitate treatment also resulted in marked elevations of foxo1 production in raw264.7 cells (fig . However, the ratio of phosphorylated versus total foxo1 levels was significantly reduced, reflecting an impaired ability of insulin to promote foxo1 phosphorylation in lps- or palmitate - treated raw264.7 cells (fig . Raw264.7 in six - well dishes were treated with either 100 ng / ml lps or 0.2 mmol / l palmitate . Aliquots of cell lyates were immunoprecipitated by anti - irs2 antibody, followed by immunoblot assay for the determination of phosphorylated irs2 using antiphosphotyrosine antibody (a). In addition, aliquots of cell lysates were subjected to immunoblot analysis for the determination of phosphorylated and total foxo1 in response to palmitate (b) or lps treatment (c). Our findings of foxo1-stimulated il-1 production in inflammation - inflicted macrophages pose two potential mechanisms: 1) foxo1 exerts an independent effect on il-1 production, and 2) alternatively, foxo1 promotes il-1 overproduction through nf-b, a well - characterized pathway that is responsible for mediating proinflammatory cytokine production in macrophages under inflammatory or insulin - resistant conditions . To distinguish these two possibilities, we chose to study the impact of foxo1 in nonstimulated macrophages . Because of a concomitant induction of endogenous foxo1 and nf-b production in lps- or palmitate - treated macrophages, unstimulated raw264.7 cells were transduced with foxo1 or control vector to determine the net effect of foxo1 on il-1 expression in the absence of nf-b activation . Unexpectedly, foxo1 failed to stimulate il-1 production, as the amount of immunoreactive il-1 protein remained unchanged in foxo1 versus control vector treated raw264.7 cells (fig . Adenovirus - mediated foxo1 production resulted in significant induction of il-1 production in raw264.7 cells that were pre - exposed to lps (fig . To ascertain this finding, we transfected phd386 encoding the il-1 promoter - directed luciferase reporter system into nonstimulated raw264.7 cells . No significant induction of luciferase activities in raw264.7 cells was detected in basal states (fig . 7b), despite a threefold elevation in foxo1 protein levels in nave raw264.7 cells that were pretransduced with foxo1 vector (fig . After 24-h incubation in the absence of lps, the conditioned medium was collected for the determination of il-1 concentrations . After 24-h incubation in the absence of lps, the relative luciferase activity in cells was determined using -galactosidase for the normalization of transfection efficiency . Cells from the experiment in b were subjected to immunoblot analysis for the determination of nuclear foxo1 protein levels . Directed luciferase reporter in the presence of foxo1 plus p50 or p65 production, using the dual luciferase system . After 24-h incubation in the absence of lps, the relative promoter activity, defined as the ratio of firefly and renilla luciferase activities, was determined . Raw264.7 cells were incubated in the absence and presence of lps (100 ng / ml) for 24 h, followed by immunoblot analysis using anti - foxo1 and anti - actin antibodies . Raw264.7 cells were transduced with foxo1 vector in the presence or absence of p50 production . After 24-h incubation in the absence of lps, cells were fixed with 1% formaldehyde, followed by chip assay using rabbit anti - foxo1 antibody . The immunoprecipitates were subjected to semiquantitative pcr assay using primers flanking the conserved ire dna motif within the il-1 promoter . After normalizing to input dna controls, raw264.7 cells were transfected with phd386 encoding the il-1 promoter directed luciferase expression system in the presence or absence of foxo1 production, using the dual luciferase system . One subset of cells was cotransfected with sirna that is targeted to the nf-b p50 subunit . Ns, not significant . To investigate whether foxo1 contributes to il-1 production via a mechanism that is dependent on nf-b, we determined the effect of foxo1 on il-1 promoter activity in the presence and absence of vector - mediated production of p50 or p65, two subunits of nf-b . In keeping with our earlier observations (fig . 7a), adenovirus - mediated foxo1 production exerted little effects on il-1 promoter activity in nonstimulated raw264.7 cells . However, cotransfection of foxo1 along with p50, but not with p65, resulted in a significant induction of il-1 promoter activity (fig . These results suggest that foxo1 associates with p50 in stimulating il-1 expression in macrophages, or, alternatively, p50 is required for the recruitment of foxo1 to its target site to stimulate il-1 promoter activity . To address the first hypothesis we show that lps, known to stimulate nf-b activation, also promoted foxo1 production in raw264.7 cells (fig . Thus, to determine the net impact of foxo1 and p50 interaction on il-1 expression, we chose to perform the studies in unstimulated raw264.7 cells with vector - mediated production of foxo1 and p50 proteins . Foxo1-expressing raw264.7 cells were transfected with plasmid p50 encoding the human nf-b p50 subunit, followed by immunoprecipitation using anti - foxo1 or anti - p50 antibodies . We detected the presence of foxo1 or p50 in immunoprecipitates that were derived from its cognate antibody, validating the method of immunoprecipitation . However, neither foxo1 nor p50 was reciprocally coimmunoprecipitated by anti - p50 or anti - foxo1 antibodies (data not shown). These results argue against the notion that foxo1 formed a complex with p50 in promoting il-1 expression in macrophages . We then addressed the second hypothesis that foxo1 binding to its target site within the il-1 promoter depends on p50 in cells . Implicit in this hypothesis is the presence of a highly conserved nf-b p50-binding site (5-gggagcatcc-3) downstream of the ire, the characteristic dna motif that is responsible for foxo1 binding (fig . We performed chip assay on raw264.7 cells in the presence of foxo1 or p50 expression alone or in combination . The resulting immunoprecipitates were analyzed by semiquantitative pcr assay using specific primers flanking the foxo1 binding site in the il-1 promoter . 7f, positive interactions between foxo1 and il-1 promoter dna were detected in response to foxo1 or p50 production in raw264.7 cells . However, coproduction of foxo1 and p50 resulted in a synergistic effect on foxo1 binding to its target site within the il-1 promoter . This effect correlated with the costimulatory effect of foxo1 and p50 in combination on il-1 promoter activity (fig . To consolidate our findings that foxo1 signaling through p50 regulates il-1 production in macrophages, we used the sirna - mediated gene - silencing approach to knockdown p50 expression, followed by determining the ability of foxo1 to stimulate il-1 expression in lps - stimulated raw264.7 cells . 7 g, adenovirus - mediated foxo1 production resulted in a marked induction of luciferase activity, which was significantly reduced after sirna - mediated p50 knockdown in lps - stimulated raw264.7 cells . These results suggest that depletion of nf-b p50 attenuated the ability of foxo1 to induce il-1 promoter activity in macrophages . Aberrant proinflammatory cytokine production is intertwined with insulin resistance in obesity and type 2 diabetes . To decipher the molecular basis that links proinflammatory cytokine production to insulin resistance, we determined the effect of foxo1 on cytokine production in macrophages, a cell type that plays a pivotal role in mediating whole - body insulin resistance in the face of low - grade inflammation . Macrophage raw264.7 cells were transduced with adv - foxo1 or adv - null vector at a predefined moi (200 pfu / cell), followed by the determination of cytokine concentrations in conditioned medium . As shown in fig . 1, transduction of raw264.7 cells by foxo1 vector resulted in a significant induction of il-1 and il-2 production without alterations in the production of other cytokines including il-4, il-5, il-6, il-12, ifn-, tnf-, and granulocyte monocyte colony - stimulating factor . In contrast, il-10 expression was significantly downregulated in response to foxo1 production in raw264.7 cells . In the absence of lps, the expression of proinflammatory cytokines remained at low basal levels independently of foxo1 action (supplemental table s1). These data underscore the importance of foxo1 in regulating cytokine production in lps - activated macrophages . Because of the close association between abnormal production of il-1 in macrophages and the pathogenesis of insulin resistance, we focused on the delineation of the molecular basis underlying foxo1-mediated induction of il-1 in this study . Raw264.7 cells were mock - treated with pbs, or transduced with adv - foxo1 or control adv - null vector at a fixed dose (moi, 200 pfu / cell). After 24-h incubation in the presence of 100 ng / ml lps, conditioned medium was harvested for profiling the production of cytokine il-1 (a), il-2 (b), il-4 (c), il-5 (d), il-6 (e), il-10 (f), il-12 (g), ifn- (h), tnf- (i), and granulocyte monocyte colony - stimulating factor (gm - csf) (j). To investigate the potential role of foxo1 in macrophage production of il-1 in vivo, we treated balb / c mice with once - daily intraperitoneal administration of lps (4 mg / kg) for 2 days . Compared to pbs - treated control mice (n = 8), lps - treated mice (n = 8) exhibited significantly elevated plasma il-1 levels (fig . Two days after lps treatment, mice were killed and peritoneal macrophages were retrieved for real - time qrt - pcr analysis . 2b), correlating with significantly elevated il-1 mrna levels in peritoneal macrophages (fig . Male balb / c mice (8 weeks old, n = 8 per group) were treated with once - daily intraperitoneal injection of lps (4 mg / kg body weight) or mock - treated with pbs for 2 days . Aliquots of blood (50 l) were sampled from the tail vein for the determination of plasma il-1 levels (a). Two days after lps treatment, peritoneal macrophages were isolated from individual mice for the preparation of total rna, which was subjected to real - time qrt - pcr assay for the determination of foxo1 (b) and il-1 (c) mrna levels using 18s rrna as control . To determine whether foxo1 plays a role in linking insulin resistance to aberrant il-1 expression, we studied foxo1 expression in peritoneal macrophages in obese db / db mice . Because of the lack of leptin receptor, db / db mice are genetically obese, developing insulin resistance and hyperinsulinemia at weaning . Two groups of male db / db mice (n = 7, male, 10 weeks old) and age- or sex - matched heterozygous db/+ lean littermates (n = 7) were fasted for 16 h, as described (30). Peritoneal macrophages were isolated from individual mice and subjected to real - time qrt - pcr analysis . As shown in fig . 3a, foxo1 expression was significantly upregulated in peritoneal macrophages of insulin - resistant obese db / db mice . Furthermore, db / db mice were associated with significantly higher levels of il-1 mrna in peritoneal macrophages (fig . 3c), which was indicative of chronic inflammation in insulin - resistant obese db / db mice . Covariation of foxo1 and il-1 expression in peritoneal macrophages of insulin - resistant db / db mice . Peritoneal macrophages were isolated from insulin - resistant obese db / db mice (n = 7) and control db/+ littermates (n = 7). Total macrophage rna was prepared and subjected to real - time qrt - pcr assay for the determination of foxo1 (a) and il-1 (b) mrna levels . We hypothesized that the il-1 gene is a foxo1 target, and foxo1 promotes il-1 expression in macrophages in response to inflammation . To test this hypothesis, we treated raw264.7 cells with palmitate, the predominant saturated form of free fatty acid that is known to elicit low - grade inflammation . 4, this treatment resulted in significant induction of il-1 production, as reflected in markedly elevated levels of il-1 protein in conditioned medium and il-1 mrna in cells . Raw264.7 cells were incubated in culture medium in the presence of 0.2 mmol / l palmitate or bsa as control for 24 h. cells were harvested and subjected to real - time qrt - pcr assay for the determination of foxo1 (a) and il-1 (b) mrna levels . To characterize the underlying mechanism, we addressed whether foxo1 targets the il-1 promoter for trans - activation . A 2-kb dna fragment harboring the mouse il-1 promoter was cloned into the pgl3-basic luciferase reporter system (fig . The resulting plasmid phd386 encoding the il-1 promoter directed luciferase was transfected via electroporation into raw264.7 cells . Il-1 promoter activity was determined in response to adenovirus - mediated foxo1 production in the presence and absence of insulin . As shown in fig . 5b, we detected a more than sixfold induction of the il-1 promoter activity in raw264.7 cells that were pretransduced with foxo1 vector . This effect was counteracted by insulin, correlating with the ability of foxo1 to undergo insulin - dependent translocation from the nucleus to cytoplasm (25,27). Raw264.7 cells in six - well microplates were transduced with adv - foxo1 or control adv - null vectors at a fixed dose (moi, 200 pfu / cell), followed by transfection with 1-g phd386 plus 1-g pca35-lacz in the presence of 100 ng / ml lps in culture medium . After 24-h incubation in the absence or presence of insulin (300 nmol / l), cells were harvested for the determination of luciferase and -galactosidase activities . The relative luciferase activity was calculated by setting the ratio of luciferase/-galactosidase activity in control cells to 1 . Raw264.7 cells were transfected with wild - type (wt) or mutant (mt) il-1 promoter directed luciferase system in the absence or presence of adenovirus - mediated foxo1 production . After 24-h incubation in the presence of 100 ng / ml lps, cells were harvested for the determination of relative luciferase activities using -galactosidase activity as internal controls . Raw264.7 cells were cotransfected with phd386 and pca35-lacz in the absence or presence of adenovirus - mediated foxo1 production . One subset of cells was transduced with 200 moi of adv - foxo1-rnai encoding foxo1-specific rnai or control adv - scrnai encoding scrambled rnai . After 24-h incubation in the presence of 100 ng / ml lps, cells were harvested for the determination of relative luciferase activities using -galactosidase activity as internal controls . The conditioned medium from the experiment in d was used for the determination of il-1 levels . Cells obtained from the experiment in d were subjected to semiquantitative immunoblot analysis for the determination of total foxo1 protein levels, using intracellular actin protein as control . Raw264.7 cells incubated in six - well dishes in the presence of lps (100 ng / ml) for 24 h. cells were fixed with 1% formaldehyde, followed by chip assay using rabbit anti - foxo1 or control igg (rabbit anti-galactosidase). Immunoprecipitates were analyzed by pcr assay for the detection of dna, using primers flanking the consensus foxo1 binding site within the il-1 promoter ., we altered the insulin - responsive element (ire) motif within the il-1 promoter via site - directed mutagenesis (supplemental fig . The resulting mutant il-1 promoter was analyzed for its ability to mediate the stimulatory effect of foxo1 on il-1 expression . As shown in fig . 5c, alterations within the ire dna motif rendered the mutant il-1 promoter unresponsive to foxo1 production in raw264.7 cells . As control, adenovirus - mediated foxo1 production resulted in a significant induction of wild - type il-1 promoter activity (fig . To further prove that foxo1 contributed to il-1 regulation, we employed an rna interference (rnai)-mediated gene silencing approach to knockdown foxo1 expression in cultured macrophages . Raw264.7 cells were transfected with phd386 expressing the il-1 promoter - directed luciferase reporter gene, followed by transduction of adenoviral vectors expressing foxo1-specific rnai or control scrambled rnai, as described (30). After 24-h incubation, cells were subjected to luciferase activity assay . As shown in fig . 5d, rnai - mediated foxo1 knockdown resulted in abolition of foxo1-mediated induction of il- promoter activity in lps - stimulated raw264.7 cells . These results were corroborated when the conditioned medium was used for the determination of il-1 protein . This effect was abolished by rnai - mediated foxo1 knockdown, as reflected in 80% of reduction in foxo1 protein levels in raw264.7 cells that were pretransduced with foxo1-rnai vector (fig . The above results spurred the idea that foxo1 stimulates il-1 expression via direct binding to the il-1 promoter . 5a), a consensus dna motif that is responsible for binding and mediating foxo1 induction of target gene expression (25,27). Interestingly, similar foxo1 consensus sites were detected in the regulatory region of both human and rat il-1 promoters, suggesting an evolutionally conserved mechanism (supplemental fig . S2). To determine the ability of the conserved ire motif to mediate the stimulatory effect of foxo1 on il-1 promoter activity, we performed chromatin immunoprecipitation (chip) assay to analyze the potential interaction of foxo1 with il-1 promoter dna in raw264.7 cells . Because of low foxo1 expression in unstimulated raw264.7 cells, we transduced raw264.7 cells with foxo1 vector in the presence of lps (100 ng / ml) in culture medium . After 24-h incubation, cells were subjected to chip assay using rabbit anti - foxo1 antibody or control rabbit igg . The resulting immunoprecipitates were subjected to pcr analysis, using primers flanking the ire motif within the il-1 promoter . A 457-bp dna corresponding to the ire region (1,359/902 nt) of the il-1 promoter was produced from anti - foxo1 immunoprecipitates (fig . In contrast, the immunoprecipitates derived from control igg were negative in the same pcr assay . To further illustrate the role of foxo1 in linking aberrant il-1 production to insulin resistance, we determined the effect of lps or palmitate treatment on insulin signaling in cultured macrophages . Raw264.7 cells were incubated in the presence of lps (100 ng / ml) or palmitate (0.2 mmol / l) for 36 h, followed by the addition of insulin (300 nmol / l) into culture medium . The dose of insulin used in culture medium was based on our preliminary studies and data in the literature (36). Cells were harvested 45 min after insulin action for the determination of insulin - stimulated phosphorylation of insulin receptor substrates (irss) and foxo1, both of which are key signaling molecules downstream of insulin action . Compared to mock - treated controls, raw264.7 cells pretreated with lps or palmitate treatment were associated with a significant reduction in insulin - stimulated phosphorylation of irs2 (fig . Because of extremely low levels of irs1 expression in raw264.7 cells, we were unable to detect insulin - stimulated phosphorylation of irs1 . (37), who show that irs1 is undetectable and irs2 is the sole substrate downstream of insulin action in primary murine macrophages . Lps or palmitate treatment also resulted in marked elevations of foxo1 production in raw264.7 cells (fig . However, the ratio of phosphorylated versus total foxo1 levels was significantly reduced, reflecting an impaired ability of insulin to promote foxo1 phosphorylation in lps- or palmitate - treated raw264.7 cells (fig . Raw264.7 in six - well dishes were treated with either 100 ng / ml lps or 0.2 mmol / l palmitate . Aliquots of cell lyates were immunoprecipitated by anti - irs2 antibody, followed by immunoblot assay for the determination of phosphorylated irs2 using antiphosphotyrosine antibody (a). In addition, aliquots of cell lysates were subjected to immunoblot analysis for the determination of phosphorylated and total foxo1 in response to palmitate (b) or lps treatment (c). Our findings of foxo1-stimulated il-1 production in inflammation - inflicted macrophages pose two potential mechanisms: 1) foxo1 exerts an independent effect on il-1 production, and 2) alternatively, foxo1 promotes il-1 overproduction through nf-b, a well - characterized pathway that is responsible for mediating proinflammatory cytokine production in macrophages under inflammatory or insulin - resistant conditions . To distinguish these two possibilities, we chose to study the impact of foxo1 in nonstimulated macrophages . Because of a concomitant induction of endogenous foxo1 and nf-b production in lps- or palmitate - treated macrophages, unstimulated raw264.7 cells were transduced with foxo1 or control vector to determine the net effect of foxo1 on il-1 expression in the absence of nf-b activation . Unexpectedly, foxo1 failed to stimulate il-1 production, as the amount of immunoreactive il-1 protein remained unchanged in foxo1 versus control vector treated raw264.7 cells (fig ., adenovirus - mediated foxo1 production resulted in significant induction of il-1 production in raw264.7 cells that were pre - exposed to lps (fig . 1a). To ascertain this finding, we transfected phd386 encoding the il-1 promoter - directed luciferase reporter system into nonstimulated raw264.7 cells . No significant induction of luciferase activities in raw264.7 cells was detected in basal states (fig . 7b), despite a threefold elevation in foxo1 protein levels in nave raw264.7 cells that were pretransduced with foxo1 vector (fig . After 24-h incubation in the absence of lps, the conditioned medium was collected for the determination of il-1 concentrations . B: raw264.7 cells were transfected with phd386 encoding the il-1 promoter directed luciferase expression system in the presence or absence of foxo1 production . After 24-h incubation in the absence of lps, the relative luciferase activity in cells was determined using -galactosidase for the normalization of transfection efficiency . Cells from the experiment in b were subjected to immunoblot analysis for the determination of nuclear foxo1 protein levels . D: raw264.7 cells were transfected with phd386 encoding the il-1 promoter directed luciferase reporter in the presence of foxo1 plus p50 or p65 production, using the dual luciferase system . After 24-h incubation in the absence of lps, the relative promoter activity, defined as the ratio of firefly and renilla luciferase activities, was determined . Raw264.7 cells were incubated in the absence and presence of lps (100 ng / ml) for 24 h, followed by immunoblot analysis using anti - foxo1 and anti - actin antibodies . Raw264.7 cells were transduced with foxo1 vector in the presence or absence of p50 production . After 24-h incubation in the absence of lps, cells were fixed with 1% formaldehyde, followed by chip assay using rabbit anti - foxo1 antibody . The immunoprecipitates were subjected to semiquantitative pcr assay using primers flanking the conserved ire dna motif within the il-1 promoter . After normalizing to input dna controls, the amount of immunoprecipitated il-1 promoter dna fragments was compared among different conditions . Raw264.7 cells were transfected with phd386 encoding the il-1 promoter directed luciferase expression system in the presence or absence of foxo1 production, using the dual luciferase system . One subset of cells was cotransfected with sirna that is targeted to the nf-b p50 subunit . To investigate whether foxo1 contributes to il-1 production via a mechanism that is dependent on nf-b, we determined the effect of foxo1 on il-1 promoter activity in the presence and absence of vector - mediated production of p50 or p65, two subunits of nf-b . In keeping with our earlier observations (fig . 7a), adenovirus - mediated foxo1 production exerted little effects on il-1 promoter activity in nonstimulated raw264.7 cells . However, cotransfection of foxo1 along with p50, but not with p65, resulted in a significant induction of il-1 promoter activity (fig . These results suggest that foxo1 associates with p50 in stimulating il-1 expression in macrophages, or, alternatively, p50 is required for the recruitment of foxo1 to its target site to stimulate il-1 promoter activity . To address the first hypothesis we show that lps, known to stimulate nf-b activation, also promoted foxo1 production in raw264.7 cells (fig . Thus, to determine the net impact of foxo1 and p50 interaction on il-1 expression, we chose to perform the studies in unstimulated raw264.7 cells with vector - mediated production of foxo1 and p50 proteins . Foxo1-expressing raw264.7 cells were transfected with plasmid p50 encoding the human nf-b p50 subunit, followed by immunoprecipitation using anti - foxo1 or anti - p50 antibodies . We detected the presence of foxo1 or p50 in immunoprecipitates that were derived from its cognate antibody, validating the method of immunoprecipitation . However, neither foxo1 nor p50 was reciprocally coimmunoprecipitated by anti - p50 or anti - foxo1 antibodies (data not shown). These results argue against the notion that foxo1 formed a complex with p50 in promoting il-1 expression in macrophages . We then addressed the second hypothesis that foxo1 binding to its target site within the il-1 promoter depends on p50 in cells . Implicit in this hypothesis is the presence of a highly conserved nf-b p50-binding site (5-gggagcatcc-3) downstream of the ire, the characteristic dna motif that is responsible for foxo1 binding (fig . We performed chip assay on raw264.7 cells in the presence of foxo1 or p50 expression alone or in combination . The resulting immunoprecipitates were analyzed by semiquantitative pcr assay using specific primers flanking the foxo1 binding site in the il-1 promoter . 7f, positive interactions between foxo1 and il-1 promoter dna were detected in response to foxo1 or p50 production in raw264.7 cells . However, coproduction of foxo1 and p50 resulted in a synergistic effect on foxo1 binding to its target site within the il-1 promoter . This effect correlated with the costimulatory effect of foxo1 and p50 in combination on il-1 promoter activity (fig . To consolidate our findings that foxo1 signaling through p50 regulates il-1 production in macrophages, we used the sirna - mediated gene - silencing approach to knockdown p50 expression, followed by determining the ability of foxo1 to stimulate il-1 expression in lps - stimulated raw264.7 cells . 7 g, adenovirus - mediated foxo1 production resulted in a marked induction of luciferase activity, which was significantly reduced after sirna - mediated p50 knockdown in lps - stimulated raw264.7 cells . These results suggest that depletion of nf-b p50 attenuated the ability of foxo1 to induce il-1 promoter activity in macrophages . Our goal is to understand the molecular basis that links insulin resistance to abnormal production of proinflammatory cytokine il-1 in macrophages . This effect was inhibited by insulin, which is correlated with the ability of foxo1 to undergo insulin - dependent phosphorylation and nuclear exclusion . Foxo1 was shown to bind to the il-1 promoter and enhance il-1 promoter activity . In accordance with these observations, there is a highly conserved foxo1 binding site within the il-1 promoter of humans, rats, and mice . Mutations of the foxo1 consensus site abrogated foxo1-mediated induction of il-1 promoter activity . In lps- or palmitate - treated macrophages, foxo1 expression along with its nuclear localization was increased, contributing to augmented foxo1 activity and elevated il-1 production . Lps or palmitate treatment also impaired the ability of insulin to promote irs2 and foxo1 phosphorylation in cultured macrophages . These data characterize foxo1 at the interface between abnormal production of proinflammatory cytokine il-1 and the pathogenesis of insulin resistance in macrophages . Macrophage production of il-1 is governed by nf-b, a master regulator that integrates inflammatory signals to cytokine gene expression (11,38). In unstimulated cells, nf-b is bound by ib and is sequestered in the cytoplasm . In the presence of inflammatory stimuli, ib is phosphorylated by ib kinase and is destined for proteasome - mediated degradation . As a result, nf-b is dissociated from ib and is translocated to the nucleus for promoting target gene expression (38). Consistent with this notion is the conservation of a consensus nf-b target site within the il-1 promoter among different species (supplemental fig . S2). However, this mechanism falls short of explaining why insulin resistance provokes the induction of il-1 production in activated macrophages . To address this fundamental issue, several independent studies show that activation of the pi3 kinase - akt pathway is effective in limiting lps - stimulated inflammatory cytokine production with uncharacterized mechanisms (3941). Here we show that macrophage il-1 expression is regulated by foxo1, a key nuclear transcriptional factor that mediates the inhibitory effect of insulin on target gene expression . In the absence of insulin, foxo1 acts in the nucleus as an enhancer for promoting target gene expression, whereas in the presence of insulin, foxo1 is phosphorylated by akt / pkb, resulting in its nuclear exclusion and contributing to the inhibition of target gene expression . This phosphorylation - dependent protein trafficking mechanism is critical for insulin to regulate foxo1 transcriptional activity in cells (2527,30). Our present studies shed light on the molecular basis that couples insulin resistance to the induction of proinflammatory cytokines . It follows that in insulin - resistant states, loss of insulin inhibition of foxo1 activity consequently results in unrestrained il-1 expression in activated macrophages (fig . Consistent with this interpretation, we show that foxo1 activity was significantly increased, correlating with impaired insulin action and elevated il-1 production in lps- and palmitate - stimulated macrophages . Furthermore, addition of insulin resulted in only partial suppression of il-1 promoter activity, which is indicative of insulin resistance in lps - stimulated raw264.7 cells (fig ., primary peritoneal macrophages retrieved from lps - treated mice and insulin - resistant db / db mice were invariably associated with increased foxo1 activity, accompanied by elevated il-1 levels in plasma . Tripathy et al . (42) show that acute elevation of free fatty acid directly provokes inflammation, culminating in enhanced nf-b activity and increased proinflammatory cytokine expression in mononuclear cells in healthy individuals . These findings together with our data support the notion that circulating mononuclear cells in insulinresistant obese subjects are in a proinflammatory state (43). Indeed, treatment with thiazolidenediones, a class of insulin sensitizers for improving peripheral insulin resistance, reduces mononuclear nf-b activity and ameliorates inflammation in obese subjects (4446). Foxo1 targets at the ire dna motif of the il-1 promoter for trans - activation . Under insulin - resistant or inflammatory conditions, foxo1 activity is increased because of impaired abilities of insulin to phosphorylate and translocate foxo1 from the nucleus to cytoplasm . This effect along with the activation of the nf-b pathway synergistically promotes macrophage production of proinflammatory cytokine il-1. The evolutionally conserved ire dna motif and nf-b binding site are indicated within the promoter - proximal region . Although foxo1 targeted il-1 promoter for trans - activation, foxo1 did not elicit a significant induction of il-1 expression in cultured raw264.7 cells in the absence of inflammatory stimuli . In response to inflammatory signals such as lps or palmitate this effect was mirrored by the activation of nf-b in lps- or palmitate - activated macrophages . Interestingly, we show that foxo1 in concert with activated nf-b produced a synergistic effect on the induction of il-1 overproduction in the presence of inflammation . Together these results suggest that augmented foxo1 activity serves to amplify the action of nf-b in stimulating macrophage il-1 production under inflammatory conditions (fig . First, the productive interaction between foxo1 and il-1 promoter dna was significantly enhanced in the presence of p50 production, correlating with the costimulatory effect of foxo1 and p50 on il-1 promoter activity in cultured macrophages . Second, sirna - mediated knockdown of p50 significantly attenuated the ability of foxo1 to stimulate il-1 expression in lps - stimulated macrophages . Third, the il-1 promoter contains both foxo1 and nf-b target sites that are juxtaposed within the promoter - proximal region of the il1b gene in mice, rats, and humans (supplemental fig . S2), implying an evolutionally conserved mechanism for foxo1 signaling through the nf-b pathway in regulating proinflammatory cytokine il-1 production in macrophages in response to insulin resistance . In conclusion, we characterized the il1b gene as a foxo1 target, demonstrating that macrophage production of il-1 was stimulated by foxo1 and inhibited by insulin . Under insulin - resistant or inflammatory conditions, foxo1 activity was augmented, because of an impaired ability of insulin to phosphorylate foxo1 and promote its nuclear exclusion . This effect along with active nf-b contributed to macrophage overproduction of il-1. Although insulin resistance is associated with low - grade inflammation in obesity and type 2 diabetes, the underlying mechanism remains obscure . Our data suggest that foxo1 signaling through nf-b plays a significant role in coupling insulin resistance to proinflammatory cytokine il-1 production in obesity and type 2 diabetes.
They are known to become symptomatic as a consequence of enlargement, infection, or rupture, the latter being an exceedingly rare complication traditionally treated with open splenectomy . We herein report a unique case of a giant epidermoid splenic cyst that ruptured spontaneously and was successfully treated with the laparoscopic approach . Laparoscopic surgery may be considered an initial treatment option in cases of very large epidermoid cysts even when rupture occurs . Laparoscopic splenectomy is an established therapeutic option for the elective treatment of nonparasitic splenic cysts . We herein report a case of a ruptured giant epidermoid splenic cyst that was successfully treated with the laparoscopic method . A 32-year - old woman was urgently admitted to our department with acute onset of pain in the left upper quadrant that radiated to the left shoulder . She had no history of antecedent abdominal trauma; however, she did have a 2-month history of mild abdominal pain and left shoulder discomfort . On physical examination, tenderness in the left upper quadrant, diminished bowel sounds, and abdominal guarding were noted . Computerized tomography of the abdomen revealed a large cystic lesion, evolving from the spleen measuring 16 cm x 14 cm with features indicative of intra- and extrasplenic rupture . Also, a significant amount of free peritoneal fluid was present (figure 1). With the imaging diagnosis of ruptured splenic cyst, the patient underwent a successful laparoscopic splenectomy . Computerized tomography of the abdomen, demonstrating the giant ruptured epidermoid splenic cyst and the intraperitoneal fluid . After the induction of general anaesthesia and endotracheal intubation, the patient was placed in the full, right lateral decubitus position with the table tilted at 20 to 30 and in a moderate reverse trendelenburg position . Pneumoperitoneum was established with the open method (hassan technique) through the umbilicus up to the level of 15 mm hg . Four 10-mm trocars were subsequently inserted along the left costal margin, allowing for bimanual operative manipulations . Splenic mobilization commenced with division of the splenocolic ligament, ligation of inferior polar vessels and division and ligation of short gastric vessels by using ligasure (ussc, autosuture co, norwalk, ct). The next operative step was the hilar control, which was accomplished by multiple firings of the endogia (ussc, autosuture co, norwalk, ct) applied directly to the splenic vascular pedicle . Once the hilum was controlled, the ligamentous posterior peritoneal attachments were divided, preserving though the phrenic attachments . Subsequently, a specially designed retrieval bag was inserted through the left lower trocar site . After the entry of the spleen into the bag, the end of the bag was brought outside the abdomen through the umbilical trocar site . Histologic examination revealed multiple splenic fragments with a total weight of 160 g and dimensions of 18 cm x 14 cm x 7 cm . The bigger splenic fragment contained an empty cyst with dimensions of 9 cm x 6 cm with a fibrotic wall bearing epithelial lining (figures 2 and 3). This report describes a rare case of a giant epidermoid splenic cyst that ruptured spontaneously and was successfully treated with laparoscopic splenectomy . Nonparasitic splenic cysts are rare splenic lesions occurring in all age groups, probably resulting from a developmental anomaly, during which primitive mesothelium becomes entrapped within the splenic parenchyma cysts, in which no lining can be demonstrated . However, the absence of an epithelial lining can be a spurious observation, so failure to identify scant remnants of this lining may lead to erroneous classification of a cyst . Of the true splenic cysts, epidermoid cysts comprise 90%, whereas dermoid cysts are less common and comprise the remaining 10% . Clinically, true splenic cysts may be completely asymptomatic, constituting an incidental finding during physical or radiologic examination or manifested with minor, non - specific symptoms, such as pain and abdominal distention due to cyst enlargement . Complications including infection and rupture are extremely rare; in a recent review, the reported overall complication rate was 5.2%, involving 6 cases of cyst rupture and 4 cases of cyst infection . Therefore, our case of spontaneous rupture of the giant splenic cyst is an exceedingly rare condition . Nonoperative treatment is recommended for small cysts, up to 5 cm in diameter, if they are totally asymptomatic and the imaging characteristics are absolutely typical . When a splenic cyst is symptomatic or if the diagnosis is in doubt, operative therapy is warranted . Elective treatment options for nonparasitic splenic cysts include total splenectomy or some spleen - conserving variants, such as removal of the cyst in its entirety along with a remnant of adjoining spleen (partial splenectomy) or leaving a small remnant of the cyst still affixed to the spleen (cystectomy or splenic decapsulation or partial cystectomy). Other limited treatments, such as percutaneous catheter drainage and sclerosis, have also been described; however, they are associated with high rates of recurrence or infection and have largely been abandoned . Total splenectomy is the only method of operative treatment in the reported cases of ruptured epidermoid splenic cysts . Interestingly enough, although the application of the laparoscopic method is well documented in elective cases, it seems quite restricted in the urgent setting . Actually, no report has been published in the english literature about laparoscopic treatment of a ruptured epidermoid splenic cyst . Because our patient was hemodynamically stable, we opted for the laparoscopic approach, which was successfully completed . It seems reasonable that acute rupture of an epidermoid splenic cyst does not constitute a contraindication for laparoscopic repair per se, and laparoscopic splenectomy may be attempted as an initial treatment option.
The nshd is a socially stratified birth cohort of 2547 men and 2815 women of white european descent born during 1 wk in 1946 who have been followed with repeated data collections since then (14). Between 2006 and 2010, at 6064 yr, contact was made with 2661 (84%) of 3164 eligible study members still alive and living in england, scotland, or wales . No contact was attempted for those who had died (n = 717), were living abroad (n = 567), had previously withdrawn from the study (n = 595), or had been lost to follow - up for more than 10 yr (n = 319). Of the 2661 who provided information, 1690 (63.5%) attended one of six regional clinical research facilities; the remainder were visited at home or completed a postal questionnaire only (18). Inclusion criteria for the current analysis was men with data on birth weight and voice breaking at age 14 yr (n = 2001). The study received multi - centre research ethics committee approval, and informed consent was given by cohort participants . Birth weight to the nearest quarter pound (113 g) was extracted from medical records and converted to kilograms . Heights and weights were measured using standard protocols at ages 2, 4, 6, 7, 11, 15, 36, 43, 53, and 6064 yr and were self - reported at ages 20 and 26 yr . Pubertal characteristics were described by the child's school doctor (medically qualified practitioners who regularly attended schools as part of routine primary care), at a special clinic examination in 1961 when the boys were 14 yr old (mean 14.5, range 14.315.2 yr). In boys, the school doctor was asked to rate appearance of the genitals as infantile, early, or advanced . Axillary hair was rated as no or yes . Voice - breaking status was rated as no, starting to break, or completely broken . During clinic visits at 6064 yr, measures of body composition were obtained in the supine position using a qdr 4500 discovery scanner (hologic inc ., bedford, ma); the same software (hologic apex version 3.1), hardware, and procedures were used in each clinic, and all measures were calibrated internally and across clinics using a european spine phantom . From these scans, whole - body fat and lean mass, android (abdominal) region fat mass and gynoid region fat mass, and appendicular lean mass were obtained and converted into kilograms . The ratio of android to gynoid fat mass was derived, with higher values indicating greater fat distribution in the abdomen than hips . Lean mass was defined as mass excluding fat and bone mass, and in all measures, data from the head were excluded due to the high proportion of bone mass known to affect the accuracy of soft - tissue measures (19). Routine anthropometric measures were taken during the clinic visit using standardized protocols by trained nurses (18). In total, 746 male participants had data available for all body composition outcomes, with missing data mostly due to artifacts such as pacemakers obstructing the x - rays . Bmi at each measurement and maternal bmi were calculated as weight in kilograms divided by height in meters squared . Measures of bmi, weight, and height were converted into sds using internally generated growth charts, which is preferable for historical cohorts, and were constructed using the lms method (where l is skewness, m is median, and s is coefficient) (20). Hd - sds was originally described as the difference between adolescent height sds (at age 14 yr in males) and adult height sds (16), and we calculated this parameter as height sds at 36 yr minus height sds at 14 yr because 36 yr was the first time adult height was measured in the nshd . Lower hd - sds values indicate those males with less growth occurring after age 14 yr and who therefore had earlier pubertal growth acceleration and more advanced pubertal maturation at age 14 yr than those with higher hd - sds values (16). Conditional hd - sds was calculated as adolescent height sds (r adult height sds)/(1 r), where r is the pearson correlation coefficient for the association between adolescent height sds and adult height sds . This equation was as previously described for conditional weight gain (17), but we arranged the variables in the equation to design a parameter that is independent of adult height . Note that higher conditional hd - sds values indicate males with more advanced puberty at age 14 yr . Linear regression was initially performed to test the cross - sectional linear trends in body size across groups of voice breaking at age 14 yr, adjusted for precise age at pubertal assessment . Random intercept mixed models were used to test the longitudinal associations between voice - breaking status at age 14 yr and changes in weight, bmi, and height sds with age, using the xtmixed command in stata . This approach takes correlations between repeated measures on the same individual into account and allows for missing measurement data assuming that data are missing at random . The linear change in outcome with age was allowed to vary by voice - breaking status by adding a voice - breaking group by age interaction term . We used wald test statistics to compare models that included linear, quadratic, and cubic functions of age to test whether changes in body size by voice - breaking status were nonlinear between birth to 6064 yr . To estimate linear coefficients between growth and age during specific developmental periods, we fitted linear piecewise models with knots at 2 (weight only), 7, 14, and 20 yr to allow different linear coefficients for 02 yr (infancy; data available for weight only), 27 yr (prepubertal childhood), 714 yr (early adolescence), 1420 yr (late adolescence), and 20 to 6064 yr (adult). The nshd is a socially stratified birth cohort of 2547 men and 2815 women of white european descent born during 1 wk in 1946 who have been followed with repeated data collections since then (14). Between 2006 and 2010, at 6064 yr, contact was made with 2661 (84%) of 3164 eligible study members still alive and living in england, scotland, or wales . No contact was attempted for those who had died (n = 717), were living abroad (n = 567), had previously withdrawn from the study (n = 595), or had been lost to follow - up for more than 10 yr (n = 319). Of the 2661 who provided information, 1690 (63.5%) attended one of six regional clinical research facilities; the remainder were visited at home or completed a postal questionnaire only (18). Inclusion criteria for the current analysis was men with data on birth weight and voice breaking at age 14 yr (n = 2001). The study received multi - centre research ethics committee approval, and informed consent was given by cohort participants . Birth weight to the nearest quarter pound (113 g) was extracted from medical records and converted to kilograms . Heights and weights were measured using standard protocols at ages 2, 4, 6, 7, 11, 15, 36, 43, 53, and 6064 yr and were self - reported at ages 20 and 26 yr . Pubertal characteristics were described by the child's school doctor (medically qualified practitioners who regularly attended schools as part of routine primary care), at a special clinic examination in 1961 when the boys were 14 yr old (mean 14.5, range 14.315.2 yr). In boys, the school doctor was asked to rate appearance of the genitals as infantile, early, or advanced . Axillary hair was rated as no or yes . Voice - breaking status was rated as no, starting to break, or completely broken . During clinic visits at 6064 yr, measures of body composition were obtained in the supine position using a qdr 4500 discovery scanner (hologic inc ., bedford, ma); the same software (hologic apex version 3.1), hardware, and procedures were used in each clinic, and all measures were calibrated internally and across clinics using a european spine phantom . From these scans, whole - body fat and lean mass, android (abdominal) region fat mass and gynoid region fat mass, and appendicular lean mass were obtained and converted into kilograms . The ratio of android to gynoid fat mass was derived, with higher values indicating greater fat distribution in the abdomen than hips . Lean mass was defined as mass excluding fat and bone mass, and in all measures, data from the head were excluded due to the high proportion of bone mass known to affect the accuracy of soft - tissue measures (19). Routine anthropometric measures were taken during the clinic visit using standardized protocols by trained nurses (18). In total, 746 male participants had data available for all body composition outcomes, with missing data mostly due to artifacts such as pacemakers obstructing the x - rays . Bmi at each measurement and maternal bmi were calculated as weight in kilograms divided by height in meters squared . Measures of bmi, weight, and height were converted into sds using internally generated growth charts, which is preferable for historical cohorts, and were constructed using the lms method (where l is skewness, m is median, and s is coefficient) (20). Hd - sds was originally described as the difference between adolescent height sds (at age 14 yr in males) and adult height sds (16), and we calculated this parameter as height sds at 36 yr minus height sds at 14 yr because 36 yr was the first time adult height was measured in the nshd . Lower hd - sds values indicate those males with less growth occurring after age 14 yr and who therefore had earlier pubertal growth acceleration and more advanced pubertal maturation at age 14 yr than those with higher hd - sds values (16). Conditional hd - sds was calculated as adolescent height sds (r adult height sds)/(1 r), where r is the pearson correlation coefficient for the association between adolescent height sds and adult height sds . This equation was as previously described for conditional weight gain (17), but we arranged the variables in the equation to design a parameter that is independent of adult height . Note that higher conditional hd - sds values indicate males with more advanced puberty at age 14 yr . Linear regression was initially performed to test the cross - sectional linear trends in body size across groups of voice breaking at age 14 yr, adjusted for precise age at pubertal assessment . Random intercept mixed models were used to test the longitudinal associations between voice - breaking status at age 14 yr and changes in weight, bmi, and height sds with age, using the xtmixed command in stata . This approach takes correlations between repeated measures on the same individual into account and allows for missing measurement data assuming that data are missing at random . The linear change in outcome with age was allowed to vary by voice - breaking status by adding a voice - breaking group by age interaction term . We used wald test statistics to compare models that included linear, quadratic, and cubic functions of age to test whether changes in body size by voice - breaking status were nonlinear between birth to 6064 yr . To estimate linear coefficients between growth and age during specific developmental periods, we fitted linear piecewise models with knots at 2 (weight only), 7, 14, and 20 yr to allow different linear coefficients for 02 yr (infancy; data available for weight only), 27 yr (prepubertal childhood), 714 yr (early adolescence), 1420 yr (late adolescence), and 20 to 6064 yr (adult). At age 14 yr, 537 (26.8%) of 2001 males had no voice breaking, 743 (37.1%) were starting, and 721 (36.0%) had complete voice breaking . Age at the time of pubertal assessment was slightly later in those with complete (14.57 yr) vs. starting (14.54) or no voice breaking (14.50; p trend <0.0001), and this was adjusted for in all subsequent analyses . Males with different voice - breaking status at age 14 yr showed divergent patterns of growth in weight, height, and bmi during childhood and adulthood (fig . 1); longitudinal models confirmed nonlinear associations with age (quadratic age terms in models for weight, height, and bmi sds were all p <0.001, not shown). Mean height, weight, and bmi sds from birth to age 6064 yr by voice - breaking status at age 14 yr . There was no difference in birth weight between groups of voice - breaking status (p trend = 0.7; table 1); thereafter, the groups diverged in weight (fig . More advanced voice breaking at age 14 yr was associated with heavier weight and bmi at age 2 yr and with a trend to taller height (table 1). In longitudinal analyses, compared with males with no voice breaking, those with starting to break (p = 0.04) and complete voice breaking at age 14 yr (p <0.0005) showed faster weight gain between birth and 2 yr (table 2). Childhood and adult weight, height, and bmi in males by voice - breaking status at age 14 yr results are shown as means, 95% confidence interval . Longitudinal analyses of voice - breaking status at 14 yr and growth and weight gain regression coefficients se, estimated from random intercept mixed models with piecewise specification of age periods, represent changes in sds per year during the specific age periods for males in the starting to break or complete voice breaking groups compared with those with no voice breaking as the reference group . Males with complete or starting voice breaking at 14 yr showed faster gains in weight and also in height during childhood compared with those with no voice breaking, during both prepubertal childhood (27 yr) and early adolescence (714 yr) (table 2 and fig . Differences in bmi were maintained during the prepubertal years (27 yr; table 1) and widened further during early adolescence (714 yr; table 2). Differences in weight, height, and bmi were all maximal at age 14 yr and reduced during late adolescence (1420 yr; tables 1 and 2 and fig . Differences in weight and bmi, but not height, by voice - breaking status at age 14 yr were apparent at all time points between 20 and 6064 yr, with only modest attenuation in the association with weight with increasing age (table 2). At age 6064 yr, when body composition was assessed by dual - energy x - ray absorptiometry (dxa), more advanced voice breaking at age 14 yr was associated with greater whole - body lean body mass (p = 0.002) and a trend toward greater appendicular lean mass (p = 0.07; table 3). Males with more advanced voice breaking at age 14 yr also showed a trend toward greater whole - body fat mass (p = 0.1), specifically greater android fat mass (p = 0.02), and a greater android to gynoid fat mass ratio (p = 0.02); they also had greater waist circumferences at age 53 yr (p = 0.002; table 3). Body composition outcomes at 6064 yr by voice - breaking status at 14 yr results are shown as means, 95% confidence interval; n = 624 for all variables . P values for linear trend were adjusted for age at pubertal assessment and height at 6064 yr . There were moderate intercorrelations between voice - breaking status and other markers of pubertal status at age 14 yr, with r values for linear associations ranging from 22.132.7% (table 4). All markers of pubertal maturation at age 14 yr showed consistent associations with childhood weight and growth, with the exception of hd - sds, which was the only marker of pubertal maturation that was associated with birth weight and adult height (table 5). Lower hd - sds values, which indicate earlier pubertal maturation, were associated with lower birth weight and shorter adult height, and hd - sds also showed different patterns of association with childhood size compared with the other markers (table 5). Intercorrelations between voice breaking and other markers of pubertal maturation at age 14 yr values are r from linear associations between each pair of variables . Hd - sds is difference in height sds between age 14 yr and adult (continuous variable). Comparison of associations between different markers of pubertal maturation at age 14 yr and childhood and adult size hd - sds is the difference in height sds between age 14 yr and adult (lower hd - sds values indicate more advanced pubertal maturation at age 14 yr). Conditional hd - sds is the conditional difference in height sds between adult and age 14 yr (higher hd - sds values indicate more advanced pubertal maturation at age 14 yr). Our new parameter, conditional hd - sds, designed to correct the bias inherent in (unconditional) hd - sds (17), showed a pattern of associations with childhood and adult weight and height that was more consistent with the other physical markers of pubertal maturation at age 14 yr (table 5). There was no difference in birth weight between groups of voice - breaking status (p trend = 0.7; table 1); thereafter, the groups diverged in weight (fig . More advanced voice breaking at age 14 yr was associated with heavier weight and bmi at age 2 yr and with a trend to taller height (table 1). In longitudinal analyses, compared with males with no voice breaking, those with starting to break (p = 0.04) and complete voice breaking at age 14 yr (p <0.0005) showed faster weight gain between birth and 2 yr (table 2). Childhood and adult weight, height, and bmi in males by voice - breaking status at age 14 yr results are shown as means, 95% confidence interval . Longitudinal analyses of voice - breaking status at 14 yr and growth and weight gain regression coefficients se, estimated from random intercept mixed models with piecewise specification of age periods, represent changes in sds per year during the specific age periods for males in the starting to break or complete voice breaking groups compared with those with no voice breaking as the reference group . Males with complete or starting voice breaking at 14 yr showed faster gains in weight and also in height during childhood compared with those with no voice breaking, during both prepubertal childhood (27 yr) and early adolescence (714 yr) (table 2 and fig . 1). Differences in bmi were maintained during the prepubertal years (27 yr; table 1) and widened further during early adolescence (714 yr; table 2). Differences in weight, height, and bmi were all maximal at age 14 yr and reduced during late adolescence (1420 yr; tables 1 and 2 and fig . Differences in weight and bmi, but not height, by voice - breaking status at age 14 yr were apparent at all time points between 20 and 6064 yr, with only modest attenuation in the association with weight with increasing age (table 2). At age 6064 yr, when body composition was assessed by dual - energy x - ray absorptiometry (dxa), more advanced voice breaking at age 14 yr was associated with greater whole - body lean body mass (p = 0.002) and a trend toward greater appendicular lean mass (p = 0.07; table 3). Males with more advanced voice breaking at age 14 yr also showed a trend toward greater whole - body fat mass (p = 0.1), specifically greater android fat mass (p = 0.02), and a greater android to gynoid fat mass ratio (p = 0.02); they also had greater waist circumferences at age 53 yr (p = 0.002; table 3). Body composition outcomes at 6064 yr by voice - breaking status at 14 yr results are shown as means, 95% confidence interval; n = 624 for all variables . P values for linear trend were adjusted for age at pubertal assessment and height at 6064 yr . There were moderate intercorrelations between voice - breaking status and other markers of pubertal status at age 14 yr, with r values for linear associations ranging from 22.132.7% (table 4). All markers of pubertal maturation at age 14 yr showed consistent associations with childhood weight and growth, with the exception of hd - sds, which was the only marker of pubertal maturation that was associated with birth weight and adult height (table 5). Lower hd - sds values, which indicate earlier pubertal maturation, were associated with lower birth weight and shorter adult height, and hd - sds also showed different patterns of association with childhood size compared with the other markers (table 5). Intercorrelations between voice breaking and other markers of pubertal maturation at age 14 yr values are r from linear associations between each pair of variables . Hd - sds is difference in height sds between age 14 yr and adult (continuous variable). Comparison of associations between different markers of pubertal maturation at age 14 yr and childhood and adult size hd - sds is the difference in height sds between age 14 yr and adult (lower hd - sds values indicate more advanced pubertal maturation at age 14 yr). Conditional hd - sds is the conditional difference in height sds between adult and age 14 yr (higher hd - sds values indicate more advanced pubertal maturation at age 14 yr). Our new parameter, conditional hd - sds, designed to correct the bias inherent in (unconditional) hd - sds (17), showed a pattern of associations with childhood and adult weight and height that was more consistent with the other physical markers of pubertal maturation at age 14 yr (table 5). Similar to females with earlier menarche, the trajectory to earlier sexual maturation in males, as indicated by earlier voice breaking, is manifested by faster weight gain and growth in infancy and early childhood and leads to persistently higher adult bmi . Unlike findings in females, timing of sexual maturation in males was not associated with birth weight or with adult height (9). It is well established that earlier pubertal maturation is linked to a faster tempo of growth, that is to say a faster growth trajectory that approaches the adult height target at an earlier age . Such differences in tempo are particularly marked during adolescence and are consequent to the pubertal rise in sex steroids (21). Our current findings support the concept that the trajectory to earlier sexual maturation in males, as well as in females, is manifested as a faster tempo of growth throughout infancy and childhood, rather than being confined to the pubertal years . Such early life processes could potentially include the transient pituitary - gonadal activation that has been described in both male (22) and female (23) infants . Alternatively, it could reflect mechanisms independent of sex hormones, such lin28 and lin28b, regulators of microrna preprocessing that have been linked to childhood growth and pubertal timing in human genetics studies (15, 24) and in mouse models (25). Males with earlier voice breaking had higher bmi than other males, and this was already apparent from ages 2 yr onward . Higher childhood body weight and bmi could also directly promote earlier pubertal onset and progression through leptin, insulin, and/or other hormonal mechanisms (26). In adult life, males with earlier voice breaking continued to have higher bmi than other males throughout the duration of follow - up with no detectable attenuation up to age 6064 yr . The differences in bmi at age 6064 yr by timing of voice breaking were accompanied by differences in whole - body lean mass and in regional fat mass, such that males with earlier voice breaking had greater total lean mass and greater abdominal fat mass . In females, puberty is associated with a rise in percent body fat, relative to body weight, from around 2329% between early to late puberty (27), and females with earlier timing of menarche have greater fat mass in late adolescence (27) and as adults (28). In contrast, puberty in males is characterized by greater gains in fat - free mass than in fat mass (29) and by greater gains in central fat relative to total body fat (30); we now show that these pubertal changes appear to be exacerbated in those males with earlier timing of sexual maturation . Our findings are consistent with data from the few other studies in males, which invariably estimated the timing of puberty by analysis of adolescent height growth . Earlier age at peak height velocity has been associated with greater central but not peripheral fat mass by dxa at age 18 yr (31); greater central to peripheral skinfold ratio at age 30 yr (32); greater bmi, waist circumference, and percent body fat at age 2735 yr (33); and higher bmi at age 63 yr (34). Only one other study used a non - growth - based measure of pubertal timing; in the 1958 british birth cohort, more advanced axillary hair stage at age 16 yr was associated with greater bmi at 23 and 33 yr old (35). Elevated lifetime bmi and adult abdominal fat in males with earlier voice breaking may have important implications for increased cvd and type 2 diabetes risks, although conversely, their greater lean body mass may be protective . In the nshd, we previously reported that male early developers, defined by a composite of pubertal measures at age 14 yr, had substantially higher blood pressure (bp) at age 53 yr; mean systolic bp was 6.4 mm hg higher in the earliest puberty group compared with the latest, and for diastolic bp, the difference was 4.6 mm hg (9). Other studies have associated earlier age at peak height velocity to higher fasting plasma triglyceride and insulin levels (33). Earlier pubertal maturation might also have adverse consequences for insulin sensitivity and glucose metabolism through mechanisms other than bmi and body composition, such as the regulation of microrna (36). Additional studies are needed to confirm these differences, to understand their mechanisms and to identify their consequences for disease and mortality as have been reported in women with early menarche (7). A key limitation in characterizing such associations in males is the difficulty in accurately assessing the timing of puberty in cohort studies . Although our findings show that the timing of voice breaking appears to be a noninvasive marker, additional studies should assess the risks of bias in observed, self - reported, or even recalled timing of voice breaking . Alternatively, where archival data are available, the difference between height at age 14 yr and adult height may provide a reasonable proxy (16). However, we found that when using hd - sds, the early maturers showed lower birth weights and lower adult heights, which was at odds with our direct markers of puberty (table 5). Our earlier analysis in nshd also reported no association between pubertal timing and adult height in men, although earlier puberty was associated with greater trunk to leg length ratio (9). It is therefore highly likely that the positive association between hd - sds and adult height is spurious due to regression to the mean inherent in the formula (hd - sds = adult height sds adolescent height sds) and that this bias led to a spurious association with birth weight and masked the true differences in infancy and early childhood size . We propose a simple modification of hd - sds to make this value independent of adult height . Conditional change in sds was originally described for weight gain from birth where it was designed to make that value independent of birth weight (17). We confirmed that conditional hd - sds was indeed independent of adult height and shows a similar pattern of associations with weight and height with the direct markers of pubertal timing (table 5). However, in females, in whom earlier timing of puberty is associated with modest reductions in adult height (7), neither hd - sds (which is strongly related to adult height) nor conditional hd - sds (which is unrelated to adult height) may be appropriate alternatives for pubertal timing, and the best noninvasive measure remains age at menarche . Strengths of our study include the direct assessment of pubertal timing during adolescence by medically qualified school doctors, combined with unique very - long - term follow - up over the life course and adult body composition assessed by whole - body dxa . Pubertal maturation was assessed by a large number of school doctors working in routine practice throughout england, scotland, and wales in 1961, before publication of the current tanner stage classification (11), and the categories used provided relatively limited discrimination between groups . However, these categories still provided sufficient power to detect highly significant associations with childhood and adult growth and weight gain, with strong consistency in these associations between the various measures of pubertal maturation . Adult height was not measured until age 36 yr, and attrition in follow - up meant that available numbers were smaller for hd - sds than for other markers of pubertal timing; however, highly significant associations were still observed . Unfortunately, body composition was not assessed before age 6064 yr of age, and it would be interesting to monitor the effects of pubertal timing on adult changes in body composition with aging . Finally, although the nshd cohort remains broadly representative of the national population of a similar age (37), in both males and females, there has been a secular trend to earlier puberty in more recent birth cohorts (38). In conclusion, similar to females with earlier menarche, the trajectory to earlier sexual maturation in males is manifested by faster weight gain from birth and faster growth from early childhood and leads to higher adult bmi, greater lean mass, and greater abdominal fat mass, with potential relevance for adult health . In studies where direct assessments of pubertal development are not available, conditional
Peptide amphiphiles are a fascinating class of bioactive molecules in which one or more lipid chains are attached to peptides, either bioderived, bioinspired, or designed . The lipid chains drive self - assembly, leading to nanostructures in which the functional peptide unit can be presented at high density, for example, on the surface of nanofibrils . The use of enzymes to modulate the self - assembly of peptide amphiphiles (pas) and other amphiphilic biomolecules such as amphiphilic peptides and peptide polymer conjugates has attracted considerable attention due to potential applications in biomedicine as well as catalysis and sensing . Enzymes have been evolved by nature to perform many essential functions in biological systems . They have excellent specificity and activity under mild environmental conditions associated with living organisms . The topic of enzyme - controlled peptide self - assembly has been the subject of a recent comprehensive review . Other reviews cover enzyme - driven (biocatalyzed) self - assembly of peptide derivatives, leading specifically to hydrogelation and the influence of enzymes on the aggregation of polymer - peptide conjugates . As well as enzyme - stimulated transitions in nanostructure, peptide systems can also undergo enzyme - mediated disassembly and enzyme - triggered self - assembly . By design of the peptide substrate, responsiveness to a wide variety of enzymes we have demonstrated a concept whereby pegylated peptide micelles prepared from an amyloid -peptide derived heptapeptide containing a diphenylalanine (ff) sequence attached to peg were enzymatically degraded by -chymotrypsin which selectively cleaves peptide bonds between aromatic residues . This released a hexapeptide (containing a c - terminal f) along with peg with an n - terminal f residue . This proof - of - principle work established a possible route to create an enzyme - responsive delivery system based on peg - coated particles which have relevance to biological applications since peg provides a sterically stabilized corona, enabling enhanced in vivo stability . Here, we investigate the influence of the model serine protease -chymotrypsin on the self - assembly of the pa c16-kkffvlk . The peptide amphiphile (pa) has been designed based on the klvff core motif from the amyloid beta (a) peptide . This contains two f residues at the c terminus which drive self - assembly through hydrophobic and -stacking interactions . Two additional lysine residues are incorporated between the hexadecyl lipid chain and the ffklvff motif to promote solubility and impart amphiphilicity . The serine protease -chymotrypsin is expected to cleave preferentially between the two f residues, although as discussed below an additional cleavage site is also identified . The pas corresponding to cleaved products c16-kkf and c16-kkff are shown to self - assemble into spherical micelles, quite distinct from the nanotubes / helical ribbons formed by c16-kkffvlk . We thus show that it is possible to develop enzyme - responsive pa systems with pronounced nanostructure transitions, leading in turn to macroscopic changes in the sample transparency . Peptide amphiphile c16-kkffvlk was purchased from cs bio (menlo park, ca). For the first batch, the molar mass by electrospray - ionization mass spectrometry was 1146.97 da (expected 1147.57 da). The molar mass by es - msi was 1147.65 da and the purity was 99.73% by hplc . Peptide amphiphile c16-kkff was synthesized by cs bio (menlo park, ca) and supplied as a tfa salt . The molecular weight was found to be 807.31 da (expected 807.11 da) as revealed by electrospray - ionization mass spectrometry . Purity of the sample was 99.88% by analytical hplc in a 0.1% tfa water / acetonitirile gradient . Peptide amphiphile c16-kkf was purchased from cs bio (menlo park, ca) and supplied as a tfa salt . Electrospray - ionization mass spectrometry revealed a molecular weight of 659.73 da (expected 659.94 da). Purity was 99.35% by analytical hplc in a 0.1% tfa water / acetonitirile gradient and supplied as a tfa salt . Peptide fvlk was custom synthesized by peptide synthetics (peptide protein research ltd . The molecular weight was found to be 505.641 da by electrospray - ionization mass spectrometry . The sample was purified in acetonitrile and water containing 0.1% tfa prior to lyophilization and was supplied as a tfa salt . The sample was purified in acetonitrile and water containing 0.1% tfa prior to lyophilization and was supplied as a tfa salt . The enzyme -chymotrypsin, which has a molecular weight of 25 kda, was purchased from sigma - aldrich (bovine pancreas extract). To characterize self - assembly in water, solutions were made by direct dissolution in ultrafiltered water, from a barnstead nanopure system . The ph of a 0.5 wt% solution of c16-kkffvlk was 4.18, and the ph was 3.84 for a 1 wt% solution . Direct - infusion electrospray mass spectra (es - ms) were obtained on a thermofisher scientific orbitrap xl instrument . The ms / ms experiments were performed with an isolated target mass of 648 da (isolation width of 3 da), and fragmentation was achieved in the hcd trap with a fragmentation energy of 35 . A mixture of 1 wt% c16-kkffvlk and 2 wt% -chymotrypsin was prepared for mass spectrometry analysis . A vial containing the solution was then placed in a sonicator at 50 c for 1 h. this temperature was found to improve the activity of -chymotrypsin (previous work showed increasing activity with temperature, but the highest temperature studied was 35 c). The pa / enzyme mixture was left to age for 4 months before mass spectrometry measurements were performed . Spectra were recorded using a nexus - ftir spectrometer equipped with a dtgs detector and a multiple reflection attenuated total reflectance (atr) system . Solutions of the pas and peptides in d2o (0.5, 1, and 2 wt%) were sandwiched in ring spacers between two caf2 plate windows (spacer 0.006 mm). Spectra were recorded with a varian cary eclipse fluorescence spectrometer with samples in 4 mm inner width quartz cuvettes . The spectra were recorded from 460 to 600 nm using an excitation wavelength ex = 440 nm . Measurements were performed on stalks prepared by drying filaments of the pas or peptides, prepared from 0.5, 1, or 2 wt% solutions . Solutions of the peptide were suspended between the ends of wax - coated capillaries and dried . The stalks were mounted (vertically) onto the four axis goniometer of a raxis iv++ x - ray diffractometer (rigaku) equipped with a rotating anode generator and a saturn 992 ccd camera . The sample the x - ray wavelength was = 1.54 . The wavenumber scale (q = 4 sin /, where 2 is the scattering angle) was geometrically calculated using the size of each pixel in the detector screen (0.0898 mm) and the sample detector distance . Saxs data were collected on the biosaxs beamline bm29 at the esrf, grenoble, france . Solutions containing 0.5, 1, or 2 wt% c16-kkffvlk were loaded in pcr tubes in an automated sample changer . Further experiments (including kinetics measurements) on a 1 wt% sample were performed on beamline id02 at the esrf, grenoble, france . Saxs data were collected with a frelon kodak ccd with a 1.2 m sample detector distance, and waxs data were measured simultaneously with an aviex ccd . Solutions were injected using a syringe into enki ki - beam thin (0.05 mm) wall 1.85 mm diameter polycarbonate capillaries which optimize background subtraction . Heat / cool experiments were performed with the sample mounted in a quartz capillary instead of the enki polycarbonate capillary . Cryo - tem was carried out using a field emission cryo - electron microscope (jeol jem-3200fsc), operating at 200 kv voltage (except for sample vlk for which the voltage was 300 kv). Images were taken in bright field mode and using zero loss energy filtering (omega type) with slit width of 20 ev . Vitrified specimens were prepared using an automated fei vitrobot device using quantifoil 3.5/1 holey carbon copper grids with the hole size of 3.5 m . Just prior to use, grids were plasma cleaned using a gatan solarus 9500 plasma cleaner and then transferred into an environmental chamber of a fei vitrobot at room temperature and 100% humidity . Thereafter, 3 l of sample solution (2 wt% concentration) was applied to the grid and it was blotted twice for 5 s and then vitrified in a 1/1 mixture of liquid ethane and propane at temperature of 180 c . The most viscous gel (5 wt% concentration) was blotted four times for 5 s. the grid with vitrified sample solution were maintained at liquid nitrogen temperature and then cryo - transferred into the microscope . Our objective was to examine the effect of -chymotrypsin (c) in cleaving the pa c16-kkffvlk and to investigate the self - assembly of the product peptides and peptide amphiphiles . Figure 1 shows an electrospray ionization mass spectrum for the pa / enzyme mixture, which reveals sharp peaks with m / z values of 660.50 and 506.33 da . These peaks are identified as c16-kkf and fvlk, respectively, since these values correspond to the expected molar masses, listed in the experimental section (i.e., ions with z = 1 are produced). These two fragments are expected to be released from c16-kkffvlk in the presence of c as the enzyme selectively catalyzes the hydrolysis of peptide bonds on the c - terminal side of phenylalanine, which in our peptide corresponds to cleavage between the two f residues . Unexpectedly we observed an additional two peaks with high abundances with m / z values of 807.57 and 359.27 da . The former is ascribed to c16-kkff and the latter as the tripeptide vlk, since the m / z values correspond to the expected molar masses for these species (again z = 1). Ms / ms fragmentation was employed to confirm the nature of the two unexpected fragments by sequentially knocking off amino acid residues from the c - terminus as shown in figure 2 . As the amino acids k then l and then v are removed from the c - terminus, a peak at 789.56 da ms / ms fragmentation indicates that c cleaves at an additional site between f and v residues and confirms the existence of the two fragments (c16-kkff and vlk). Es - ms for a mixture of 1 wt% c16-kkffvlk and 2 wt% -chymotrypsin . Ms / ms fragmentation spectrum for c16-kkffvlk, sequentially removing lysine, leucine, and valine from the c - terminus . To further elucidate the enzymatic degradation products of the pa by -chymotrypsin, figure 3 shows spectra for the pa and the enzyme alone, as well as the mixture . It is clear that the cd spectrum from the mixture is completely different from either of the two components and cannot be expressed as a superposition of the spectra from the species in the mixture . The spectrum for c alone is consistent with that previously reported by ourselves and others . The spectrum for c16-kkffvlk is dominated by electronic transitions associated with the stacking of the f residues, as discussed elsewhere . The expected cd spectrum for -sheet structures, with a negative minimum near 216 nm and a positive maximum in the 195202 nm range, we provide evidence in a recent paper that this pa does form -sheet fibrils and this is supported by further data provided herein, to be discussed shortly . Cd spectra for 1 wt% c16-kkffvlk and 1 wt% -chymotryspin and their mixture (1 and 1 wt%). Cd was also used to examine the self - assembly of peptides and pas corresponding to the cleavage products, c16-kkf and fvlk and c16-kkff and vlk, respectively . The spectra for the two pas (figure 4a, b) do not show features of canonical secondary structure such as -sheets . The spectra contain contributions arising from stacking of phenylalanine residues which as shown in our papers on phenylalanine - rich peptides and peptide conjugates can give rise to a positive maximum in the cd spectrum at 210220 nm . There is also a contribution from polyproline ii (ppii) structure in this region . The presence of a deep minimum in a cd spectrum at around 190 nm, along with a broad positive maximum at around 210 nm, is a signature of ppii (collagen - like) secondary structure . The spectra for vlk and fvlk in figure 4c and d show these features . The spectra for flvk also show features in the 240270 nm range associated with vibronic transitions of the phenylalanine chromophore . Cd spectra for solutions of 0.5, 1, and 2 wt% for (a) c16-kkff, (b) c16-kkf, (c) vlk, and (d) fvlk . Spectra covering the amide i and amide ii regions from 0.5, 1, and 2 wt% solutions of the pas and peptides are shown in figure 5 . The spectra for c16-kkffvlk show a peak at 1626 cm which is due to -sheet structure, along with a peak at 1672 cm which is due to bound tfa counterions . The spectra for c16-kkff and c16-kkf do not show peaks in the 16151635 cm range typical of -sheet structures but have broad peaks near 1641 cm . Peak positions in this range are typically ascribed to random coil structure; however, as discussed elsewhere, ppii structures can also give a peak in this region . All of the pas also exhibit broad peaks in the amide ii region centered on 1580 cm . The spectra for peptides fvlk and vlk in figure 5d, e show broad shoulder peaks in the 16401650 cm region due to random coil / ppii structure (along with the peak due to bound tfa counterions). Peaks in the amide i region are absent for the peptides indicating a lower extent of ordering of n ftir spectra for solutions of 0.5, 1, and 2 wt% for (a) c16-kkffvlk, (b) c16-kkff, (c) c16-kkf, (d) fvlk, and (e) vlk . Fiber x - ray diffraction was also used to examine peptide secondary structure . Diffraction patterns from dried stalks are shown in supporting information figure 1 along with meridional and equatorial intensity profiles . The peak positions were used to calculate d spacings and these are listed in supporting information table 1 . The pattern for c16-kkffvlk (supporting information figure 1a) shows features of a cross- structure with meridional reflections from a d = 4.83 spacing (corresponding to the -strand spacing) along with a series of equatorial reflections from the -sheet stacking distance and lateral packing of the pa molecules . A series of off - axis reflections close to the equator are due to the helical twisting of nanoribbons . The xrd pattern shows a high degree of alignment and multiple bragg peaks confirming the regular ordering of the pa within the nanotubes / ribbons . In contrast, the xrd patterns for c16-kkf and c16-kkff do not show orientation, and only present a small number of reflections . The pattern for c16-kkff indicates the lack of secondary structure for this pa since cross- features are not observed, just sharp peaks corresponding to the packing of the lipid chains along with a broad 5.7 ring due to the spacing of the molecules . Unexpectedly, c16-kkf shows more order in its xrd pattern than c16-kkff with some cross- features (i.e., 4.79/4.65 spacings and a 13.4 peak) although the 5.7 ring and the 4.11 peak from the packing of the lipid chains predominate . The self - assembled nanostructures of the pas (c16-kkffvlk and pas corresponding to the fragments produced after enzymatic degradation) and peptide fragments were examined by cryogenic transmission electron microscopy (cryo - tem). C16-kkffvlk forms nanotubes coexisting with helical ribbons (figure 6a and ref (22)). Saxs data (figure 7) confirm this assignment, as discussed in detail elsewhere . In complete contrast, c16-kkff and c16-kkf self - assemble into small (approximately 5 nm diameter) micelles . This was confirmed by saxs (figure 8) since the intensity profiles were fitted by form factors corresponding to uniform spheres, yielding radii of 2.5 and 2.9 nm for c16-kkf and c16-kkff, respectively . Cryo - tem did not reveal a significant extent of nanostructure formation for peptide fragments fvlk and vlk . There was some evidence for sparse fibril clusters for fvlk (figure 6d), and for vlk occasional raftlike film structures were observed (figure 6e). However, saxs on solutions of these two peptides (data not shown) revealed no scattering above background . The lack of significant structures for these two peptides is also consistent with ftir . The observed raftlike film structures for vlk may be due to the surface activity of this peptide . Cryo - tem images for solutions containing 1 wt% (a) c16-kkffvlk, (b) c16-kkff, (c) c16-kkf, (d) fvlk, and (e) vlk . Saxs profiles fitted to a spherical shell form factor model for (a) 0.5 wt% c16-kkf and (b) 0.5 wt% c16-kkff . The difference in morphology of the aggregates of c16-kkffvlk and the fragments c16-kkf and c16-kkff leads to changes in sample transparency as shown in figure 9 . The large nanotube / ribbon structures formed by c16-kkffvlk cause light scattering and a partly cloudy appearance, whereas the micellar structures of c16-kkf and c16-kkff show higher transparency . Confirmation that addition of enzyme to a c16-kkffvlk solution leads to a clear solution due to the cleavage process producing very small micelles is provided by the image shown for the mixture in figure 9 . Image of samples (1 wt%) in vials from left to right: c16 - kkffvlk, c16-kkff, c16-kkf, and c16-kkffvlk with added c (2 wt%). As discussed in more detail elsewhere, c16-kkffvlk undergoes a thermoreversible transition from nanotubes coexisting with helical ribbons (unwound nanotubes) at 20 c to twisted tapes at higher temperature (55 c). The oscillations in the data at 20 c are characteristic of a nanotube / ribbon bilayer structure as analyzed by form factor fitting . These disappear on heating, showing the break - up of the nanotubes / ribbons . However, on recooling, the oscillations develop slowly over a period of hours . The enzymatic cleavage of a pa using the model protease -chymotrypsin has been demonstrated . Es - ms reveals that c attacks two cleavage sites within the pa and preferentially cleaves between the two f residues as expected, leading to a higher abundance of both c16-kkf and fvlk . The second cleavage process occurs at the c terminus of the second f residue in c16-kkffvlk to produce c16-kkff and vlk . We have shown that the pas corresponding to the cleavage products c16-kkf and c16-kkff assemble into very different structures than the parent pa which forms nanotubes and helical ribbons at room temperature . In contrast to c16-kkffvlk, the two pas with shorter peptide headgroups lack substantial defined secondary structure (there were some signs of -sheet content in the xrd pattern for c16-kkf, possibly due to reduced hydrophobicity compared to c16-kkff). Indeed, a phase diagram assembled for pas on the basis of molecular dynamics computer simulations anticipates the formation of spherical micelles when hydrogen bonding interactions are weak . The near absence of intermolecular hydrogen bonded -sheets for c16-kkf or c16-kkff presumably reflects the insufficient length of the peptide headgroup, i.e. There are insufficient residues to participate in extensive hydrogen bonding networks . The change in nanostructure from c16-kkffvlk to c16-kkf and c16-kkff leads to macroscopic changes in sample appearance which might be useful for a simple enzyme sensing system, although more sensitive methods may be available . We have recently investigated the self - assembly of the pa c16-kttks which forms extended -sheet nanotapes . Considering in addition the findings reported here, our results on lysine - rich pas suggest that a minimal sequence capable of forming -sheets may require around five residues, provided that the peptide has both highly soluble hydrophilic residues along with hydrophobic residues . However, this is not a general rule since, for example, we have recently shown that a pa c16-ah with only a dipeptide -alanine - histidine headgroup can self - assemble into -sheet fibrils in water . Neither of the peptide fragments vlk nor fvlk form extensive self - assembled nanostructures, although tripeptides are able to do so provided they have sufficient amphiphilicity (e.g., if they contain two hydrophobic phenylalanine residues). Our own work also shows that a hexapeptide with one terminal phenylalanine does not self - assemble whereas a homologous heptapeptide with two c - terminal phenylalanines is capable to form -sheet fibrils . One interesting application of our lysine - rich pas may be antimicrobial agents . The antimicrobial activity of pas c16-kxk (where x is a, g, l or k or k, i.e. D - lysine) and c16-kk and c16-k has been investigated, and this was correlated to their self - assembly properties . The most active pa (against microbes) c16-kkk formed very small oligomers in contrast to c16-kgk and c16-klk which formed fibrils and c16-kak which formed 10 nm diameter micelles . Our results highlight the ability of enzymes to modulate the self - assembly of pa systems and the ability to tune nanostructure through use of enzymatic triggers . Our proof - of - concept work may be extended to create other enzyme - responsive assemblies or in applications where release of peptide or pa fragments is beneficial . Specifically, release of micelles in response to enzymatic degradation of extended nanostructures (which have been shown to offer enhanced circulation times in vivo) may be useful in delivery of actives.

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